In rodents, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) catalyzes the conversion of inactive 11-dehydrocorticosterone to the active hormone corticosterone. Dysregulation of intracellular glucocorticoid action is implicated in metabolic diseases. Assessing 11β-HSD1 enzyme levels in vivo may be key to understanding obesity pathophysiology.
We used a Zucker Fatty (ZF) rat model and [18F]AS2471907 PET imaging to determine appropriate kinetic modeling methods and assess changes in 11β-HSD1 levels due to obesity in the liver, white and brown adipose tissue (WAT/BAT), and brain.
To validate [18F]AS2471907 PET in preclinical models, time-activity curves (TACs) were generated and kinetic modeling was performed with image-derived input functions (IDIFs) extracted from multiple locations. Quantitative estimates of radioligand binding were compared with ex vivo 11β-HSD1 protein expression. Validated quantitative PET kinetic modeling methods were then used to assess differences in 11β-HSD1 between lean and obese ZF rats. Metabolic disease status was confirmed with stable isotopes tracer studies of glucose and fatty acid metabolism.
Obesity is associated with decreased brain 11β-HSD1 levels, measured by [18F]AS2471907 PET, which correlated with measures of glucose and fatty acid metabolism.
We demonstrate that [18F]AS2471907 PET can provide useful quantification of 11β-HSD1 levels in a rodent model of obesity.
© The Author(s) 2024.
Product Citations: 218
In Molecular Imaging on 24 April 2025 by Bini, J., Strober, J., et al.
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Preprint on BioRxiv : the Preprint Server for Biology on 6 February 2025 by Ma, R., Gong, L., et al.
ABSTRACT Background The liver undergoes significant hemodynamic changes during surgery, transplantation, or cirrhosis with portal hypertension(PH). The hepatic artery buffer response(HABR), which compensates for reduced portal venous flow by increasing hepatic artery(HA) flow, is hypothesized to induce pathological portal tract remodeling. This study investigates the molecular mechanisms underlying this process. Methods PH was induced in Sprague-Dawley rats via partial portal vein ligation(PPVL). Structural evaluation(microCT), immune cell profiling, hemodynamic measurements, and transcriptomic analysis in macrophages(Mϕ) from sham or PPVL rats were conducted. Results MicroCT revealed decreased portal vein flow and increased HA flow correlated with portal pressure(r=0.799, p<0.01). A 2-fold increase in portal tract fibrosis(p<0.001) was observed with increased α-SMA+ myofibroblasts in PPVL rats. CD68+ Mϕ peaked at 10 days post-PPVL, and their depletion significantly reduced fibrosis(p<0.001), indicating critical roles of Mϕ in portal tract remodeling. VCAM-1 was elevated in HA endothelium and portal fibroblasts (PFs); VCAM-1 neutralization reduced collagen accumulation(p<0.05), CD68+ Mϕ(46.3%, p<0.01), and CD3+ T cells(18%, p<0.05). Mϕ-conditioned medium increased VCAM-1 in PFs(8-fold, p<0.001) and enhanced PF migration, while VCAM-1 knockdown reduced this effect (p<0.01). Single-cell RNA sequencing data(GSE171904) and RNA-FISH revealed increased interactions between osteopontin (Spp1)+ Mϕ and PFs, with Spp1+ Mϕ driving fibrosis. Spp1 knockdown in Mϕ co-culture reduced PF fibrogenic markers, while recombinant Spp1 upregulated Col1a1, Fn1, and Acta2 expression in PFs. Conclusion Increased VCAM-1 in arterial endothelial cells and PFs facilitates the recruitment of Spp1+ Mϕ, which drive HA flow-mediated vascular remodeling and portal tract fibrosis. These findings highlight arterial flow-induced fibrosis as a key mechanism in PH, potentially contributing to disease progression and decompensation. Synopsis Liver hemodynamic changes in portal hypertension drive extracellular matrix accumulation and portal tract remodeling via Spp1+ macrophages. This study highlights how altered blood flow induces fibrosis, and its potential role in decompensation, and identifies therapeutic targets for advanced liver disease.
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Cardiovascular biology
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In Nature Communications on 1 February 2025 by Van den Bossche, V., Vignau, J., et al.
Anti-epidermal growth factor receptor (EGFR) therapy (cetuximab) shows a limited clinical benefit for patients with locally advanced or recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), due to the frequent occurrence of secondary resistance mechanisms. Here we report that cetuximab-resistant HNSCC cells display a peroxisome proliferator-activated receptor alpha (PPARα)-mediated lipid metabolism reprogramming, with increased fatty acid uptake and oxidation capacities, while glycolysis is not modified. This metabolic shift makes cetuximab-resistant HNSCC cells particularly sensitive to a pharmacological inhibition of either carnitine palmitoyltransferase 1A (CPT1A) or PPARα in 3D spheroids and tumor xenografts in mice. Importantly, the PPARα-related gene signature, in human clinical datasets, correlates with lower response to anti-EGFR therapy and poor survival in HNSCC patients, thereby validating its clinical relevance. This study points out lipid metabolism rewiring as a non-genetic resistance-causing mechanism in HNSCC that may be therapeutically targeted to overcome acquired resistance to anti-EGFR therapy.
© 2025. The Author(s).
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Homo sapiens (Human)
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Biochemistry and Molecular biology
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Cancer Research
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Cell Biology
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In Scientific Reports on 9 January 2025 by Čináková, A., Vavrincova-Yaghi, D., et al.
Oxidative stress and apoptosis are highly engaged in development of diabetic nephropathy (DN). In monotherapy, dapagliflozin and pioglitazone positively modulate target organ damage even independently of their hypoglycaemic effect. This study evaluated whether a simultaneous PPARγ activation and SGLT cotransporter inhibition offer superior protection against DN-related oxidative and apoptotic processes in a T1DM rat model. Diabetes was induced in Wistar rats using streptozotocin (55 mg/kg, i.p.). The rats received daily chow containing dapagliflozin (10 mg/kg), pioglitazone (12 mg/kg) or their combination. Six weeks after STZ administration, histological and molecular analyses were performed in excised kidneys. STZ-induced DN was demonstrated by the propagation of apoptotic (Bax, p53, Casp3) and oxidative reactions (Gp91phox, MnSOD) and disrupted nitric oxide signalling (eNOS, Hsp90, Cav1). Kidney damage molecule expression (Kim1, Nphs1) revealed a deceleration of kidney damage by pioglitazone and dapagliflozine monotherapies. The monotherapy also reduced apoptosis, oxidative stress, and partially restored NO signalling. The combined therapy ameliorated glomerulosclerosis but in other measured parameters, it reached the effect of the monotherapies except for Hsp90 expression modulation. Both dapagliflozin and pioglitazone exert protective character in kidneys when used in monotherapy. The combined therapy does not exhibit an expected additive effect within modulating oxidative stress, NO signalling or apoptosis.
© 2025. The Author(s).
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Rattus norvegicus (Rat)
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Ketogenic Diet Reduces Age-Induced Chronic Neuroinflammation in Mice.
In Aging Biology on 19 December 2024 by Nomura, M., Murad, N. F., et al.
The ketone body beta-hydroxybutyrate (BHB) is an acidic energy metabolite that is synthesized during periods of fasting or exercise. Our previous study demonstrated that an every other week cyclic ketogenic diet (Cyclic KD), which induces blood BHB levels similar to those observed during fasting, reduces midlife mortality and improves memory in aging mice. In addition to its canonical role as an energy metabolite, BHB regulates gene expression and inflammatory activation through non-energetic signaling pathways. The precise mechanisms by which BHB or KD affects brain function during aging remain incompletely understood. Using bulk RNA-sequencing (RNA-Seq), we examined whole brain gene expression of 12-month-old C57BL/6JN male mice fed KD for either one week or 14 months. While one-week KD increases some inflammatory gene expression, the 14-month Cyclic KD largely reduces age-induced neuroinflammatory gene expression. Next, a gene expression analysis of human primary brain cells (microglia, astrocytes, and neurons) using RNA-Seq revealed that BHB alone induces a mild level of inflammation in all three cell types. However, BHB inhibits the more pronounced inflammatory gene expression induced by lipopolysaccharide (LPS) in microglia. BHB exhibits a comparable inhibitory effect on LPS-induced inflammation in mouse primary microglia, which we used as an in vitro model to test and exclude known mechanisms by which BHB regulates inflammation and gene expression as responsible for this modulation of LPS-induced inflammatory gene expression. An acidic milieu resulting from BHB may be required for or contribute to the effect. Overall, we observe that BHB has the potential to attenuate the microglial response to inflammatory stimuli, such as LPS. This may contribute to an observed reduction in chronic inflammation in the brain following long-term Cyclic KD treatment in aging mice.
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In Nat Commun on 16 March 2024 by Zhao, Y., Ning, J., et al.
Fig.7.H
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Tead3 promotes osteoclastogenesis and mediates the effect of Malat1 deficiency.a Immunoblotting of Cas9 and MCP in RAW264.7 cells transduced with lenti-dCas9-VP64 and lenti-MS2-P65-HSF1. b qPCR (upper) and immunoblotting (lower) of Tead3 in RAW264.7 cells with CRISPRa-mediated overexpression of T...
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Tead3 promotes osteoclastogenesis and mediates the effect of Malat1 deficiency.a Immunoblotting of Cas9 and MCP in RAW264.7 cells transduced with lenti-dCas9-VP64 and lenti-MS2-P65-HSF1. b qPCR (upper) and immunoblotting (lower) of Tead3 in RAW264.7 cells with CRISPRa-mediated overexpression of Tead3. n = 3 biological replicates per group. c, d TRAP staining images (c) and quantification (d) of control and Tead3-overexpressing RAW264.7 cells treated with RANKL (50 ng/mL) for 5 days. Multinucleated TRAP-positive cells (outlined by dashed lines) were counted. Scale bars, 100 μm. n = 3 wells per group. e–g qPCR of Nfatc1 (e), Trap5 (f), and Ctsk (g) in control and Tead3-overexpressing RAW264.7 cells treated with RANKL (50 ng/mL) for 3 days. n = 3 biological replicates per group. h Immunoblotting of Nfatc1, Ctsk, and Hsp90 in control and Tead3-overexpressing RAW264.7 cells treated with RANKL (50 ng/mL) for 2 days and 5 days. i Immunoblotting of Tead3 and Hsp90 in RAW264.7 cells transfected with two independent Tead siRNAs or scrambled negative control (NC). j, k TRAP staining images (j) and quantification (k) of control and Tead3-knockdown RAW264.7 cells treated with RANKL (50 ng/mL) for 5 days. Multinucleated TRAP-positive cells (outlined by dashed lines) were counted. Scale bars: 100 μm. n = 3 wells per group. l Immunoblotting of Nfatc1, Ctsk, and β-actin in control and Tead3-knockdown RAW264.7 cells treated with RANKL for 3 days and 5 days. m Immunoblotting of Tead3 and Hsp90 in control and Malat1-knockdown RAW264.7 cells transfected with Tead3 siRNA or scrambled negative control (NC). n–p Control and Malat1-knockdown RAW264.7 cells were transfected with Tead3 siRNA or scrambled negative control (NC). 24 h after siRNA transfection, the cells were treated with RANKL for 5 days, followed by TRAP staining (n) and quantification (o) of multinucleated TRAP-positive cells (outlined by dashed lines). Scale bars, 100 μm. Cell lysates were subjected to immunoblotting of Nfatc1, Ctsk, and β-actin (p). n = 3 wells per group in o. Statistical significance in b, d–g, k, and o was determined by a two-tailed unpaired t test. Error bars are s.e.m. The experiments in a, h, i, l, m, and p were repeated independently three times, yielding similar results. Source data are provided as a Source Data file.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Nat Commun on 16 March 2024 by Zhao, Y., Ning, J., et al.
Fig.6.F
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Homo sapiens (Human)
Malat1 binds to Tead3 to inhibit Nfatc1 activity.a Immunoblotting of Yap and Tead1-4 in B16F1, MEF, BMM, MSC, L929, and RAW264.7 cells. b, c Total Tead (b) or Tead3 (c) was immunoprecipitated from RAW264.7 cells. Tead- or Tead3-bound Malat1 was quantitated by qPCR. d Biotinylated (Btn) Malat1 fra...
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Malat1 binds to Tead3 to inhibit Nfatc1 activity.a Immunoblotting of Yap and Tead1-4 in B16F1, MEF, BMM, MSC, L929, and RAW264.7 cells. b, c Total Tead (b) or Tead3 (c) was immunoprecipitated from RAW264.7 cells. Tead- or Tead3-bound Malat1 was quantitated by qPCR. d Biotinylated (Btn) Malat1 fragments were synthesized in vitro (upper), incubated with V5-Tead3-overexpressing HEK293T cell lysates, and pulled down with streptavidin beads. The bound proteins were immunoblotted with a V5-specific antibody (lower). e, f Immunoblotting of NFATC1, Ctsk or TRAP, and TEAD1-4 in RANKL-treated RAW264.7 (e) or U937 (f) cells. SE short exposure, LE long exposure. g HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with S-protein beads or a MYC-specific antibody and immunoblotting with antibodies against MYC and FLAG. h, i Control and MALAT1-knockout HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with a MYC-specific antibody (h) or S-protein beads (i) and immunoblotting with antibodies against MYC and FLAG. j, k MALAT1-knockout and Malat1-restored HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3, followed by pulldown with a MYC-specific antibody (j) or S-protein beads (k) and immunoblotting with antibodies against MYC and FLAG. l Mouse Nfatc1, Tead3, and truncation mutants. m HEK293T cells were co-transfected with MYC-Tead3 and SFB-Nfatc1 (full-length or truncated), followed by pulldown with S-protein beads and immunoblotting with antibodies against MYC and FLAG. n HEK293T cells were co-transfected with SFB-Nfatc1 and MYC-Tead3 (full-length or truncated), followed by pulldown with a MYC-specific antibody and immunoblotting with antibodies against MYC and FLAG. o, p Luciferase activity in HEK293T cells co-transfected with Tead3, constitutively active Nfatc1 (Nfatc1-CA), Renilla luciferase, and a firefly luciferase reporter containing tandem Nfatc1-binding sites (o) or the Ctsk promoter (p). q, r Luciferase activity in wild-type, MALAT1-knockout, and Malat1-restored HEK293T cells co-transfected with Tead3, Nfatc1-CA, Renilla luciferase, and a firefly luciferase reporter containing tandem Nfatc1-binding sites (q) or the Ctsk promoter (r). Statistical significance in b, c, and o–r was determined by a two-tailed unpaired t test. Error bars are s.e.m. n = 3 biological replicates. The experiments in a, d–k, m, and n were repeated independently three times, yielding similar results. Source data are provided as a Source Data file.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Nat Commun on 16 March 2024 by Zhao, Y., Ning, J., et al.
Fig.7.A
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Tead3 promotes osteoclastogenesis and mediates the effect of Malat1 deficiency.a Immunoblotting of Cas9 and MCP in RAW264.7 cells transduced with lenti-dCas9-VP64 and lenti-MS2-P65-HSF1. b qPCR (upper) and immunoblotting (lower) of Tead3 in RAW264.7 cells with CRISPRa-mediated overexpression of T...
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Tead3 promotes osteoclastogenesis and mediates the effect of Malat1 deficiency.a Immunoblotting of Cas9 and MCP in RAW264.7 cells transduced with lenti-dCas9-VP64 and lenti-MS2-P65-HSF1. b qPCR (upper) and immunoblotting (lower) of Tead3 in RAW264.7 cells with CRISPRa-mediated overexpression of Tead3. n = 3 biological replicates per group. c, d TRAP staining images (c) and quantification (d) of control and Tead3-overexpressing RAW264.7 cells treated with RANKL (50 ng/mL) for 5 days. Multinucleated TRAP-positive cells (outlined by dashed lines) were counted. Scale bars, 100 μm. n = 3 wells per group. e–g qPCR of Nfatc1 (e), Trap5 (f), and Ctsk (g) in control and Tead3-overexpressing RAW264.7 cells treated with RANKL (50 ng/mL) for 3 days. n = 3 biological replicates per group. h Immunoblotting of Nfatc1, Ctsk, and Hsp90 in control and Tead3-overexpressing RAW264.7 cells treated with RANKL (50 ng/mL) for 2 days and 5 days. i Immunoblotting of Tead3 and Hsp90 in RAW264.7 cells transfected with two independent Tead siRNAs or scrambled negative control (NC). j, k TRAP staining images (j) and quantification (k) of control and Tead3-knockdown RAW264.7 cells treated with RANKL (50 ng/mL) for 5 days. Multinucleated TRAP-positive cells (outlined by dashed lines) were counted. Scale bars: 100 μm. n = 3 wells per group. l Immunoblotting of Nfatc1, Ctsk, and β-actin in control and Tead3-knockdown RAW264.7 cells treated with RANKL for 3 days and 5 days. m Immunoblotting of Tead3 and Hsp90 in control and Malat1-knockdown RAW264.7 cells transfected with Tead3 siRNA or scrambled negative control (NC). n–p Control and Malat1-knockdown RAW264.7 cells were transfected with Tead3 siRNA or scrambled negative control (NC). 24 h after siRNA transfection, the cells were treated with RANKL for 5 days, followed by TRAP staining (n) and quantification (o) of multinucleated TRAP-positive cells (outlined by dashed lines). Scale bars, 100 μm. Cell lysates were subjected to immunoblotting of Nfatc1, Ctsk, and β-actin (p). n = 3 wells per group in o. Statistical significance in b, d–g, k, and o was determined by a two-tailed unpaired t test. Error bars are s.e.m. The experiments in a, h, i, l, m, and p were repeated independently three times, yielding similar results. Source data are provided as a Source Data file.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 17 July 2023 by He, Y., Zhang, Q., et al.
Fig.2.C
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ZHX2 represses E-cadherin expression in TNBC.A MCF10A cells infected with lentivirus encoding control vector or V5-ZHX2. Cells were harvested and immunoblotted with antibodies as indicated. B MDA-MB-231 cells infected with lentivirus encoding two individual ZHX2 sgRNAs (1 and 2) or control sgRNA....
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ZHX2 represses E-cadherin expression in TNBC.A MCF10A cells infected with lentivirus encoding control vector or V5-ZHX2. Cells were harvested and immunoblotted with antibodies as indicated. B MDA-MB-231 cells infected with lentivirus encoding two individual ZHX2 sgRNAs (1 and 2) or control sgRNA. Cells were harvested and immunoblotted with antibodies as indicated. C, D MDA-MB-231 C and SUM159 D cells infected with lentivirus encoding sg1-ZHX2 or control sgRNA, and rescued by ectopic expression of V5-ZHX2 in the knockout clones. Expression of epithelial cell marker (E-cadherin) and mesenchymal cell markers (Vimentin) were examined by immunoblotting with antibodies as indicated. * indicates the predicted position. E, F Immunofluorescence staining of E-cadherin (green) and Vimentin (red) in MDA-MB-231 E and SUM159 F cells. Scale bar 15 μm. Staining of DAPI (blue) indicates nuclei. G qRT-PCR analysis of CDH1 from MDA-MB-231 (upper) and SUM159 (lower) cells. Error bars represent mean ± standard deviation (SD), one-way ANOVA. **p < 0.01; ***p < 0.001; ****p < 0.0001.
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Sci Adv on 23 June 2023 by Nishimura, T., Lazzeri, G., et al.
Fig.1.D
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AHATG3 cannot be substituted for by unrelated AHs in human ATG3 protein.(A) Far ultraviolet (UV) CD spectra of AHATG3 peptide (75 μM) in the absence or presence of DOPC/DOPE (70/30) liposomes (6 mM). A peptide corresponding to the N-terminal ATG3 (1 to 22 amino acids) sequence and containing addi...
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AHATG3 cannot be substituted for by unrelated AHs in human ATG3 protein.(A) Far ultraviolet (UV) CD spectra of AHATG3 peptide (75 μM) in the absence or presence of DOPC/DOPE (70/30) liposomes (6 mM). A peptide corresponding to the N-terminal ATG3 (1 to 22 amino acids) sequence and containing additional C-terminal WK residues was used to facilitate titration by UV spectroscopy. The diameter of sonicated liposomes and liposomes extruded through 0.1- or 0.2-μm filters were 38, 100, and 147 nm, respectively. MRE, mean residue ellipticity. (B) Helicity at 222 nm as determined from the spectra shown in (A). Data represent the means ± SEM of three biological replicates. (C) An experimental scheme of ATG3 rescue assay using ATG3 chimeras. ATG3 KO MEFs expressing ATG3 WT, ATG3 ΔAH deletion mutant, or ATG3 chimera were used to analyze LC3 lipidation and LC3 puncta formation. The domain organization of ATG3-inGFP is shown: A GFP tag is inserted into the region after E125 residue. P21 and L23 in the conserved region following AHATG3 are coded in all ATG3 constructs. (D and F) LC3 flux assay. The indicated cells were starved for 6 hours with (B) or without 100 nM BafA1 (S) or cultured in full media (F). Cell lysates were analyzed by immunoblotting using the indicated antibodies. (E) LC3 puncta formation. The cells were starved for 1 hour, fixed, and stained with anti-LC3 antibody. The specimens were analyzed by an FV3000 confocal microscope. Arrowheads indicate the membrane localization of ATG3 chimeras. Scale bar, 10 μm. Differences were statistically analyzed by one-way analysis of variance (ANOVA) and Tukey multiple comparison test. ***P < 0.001.
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Collected and cropped from Sci Adv by CiteAb, provided under a CC-BY license
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In Sci Adv on 23 June 2023 by Nishimura, T., Lazzeri, G., et al.
Fig.1.F
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AHATG3 cannot be substituted for by unrelated AHs in human ATG3 protein.(A) Far ultraviolet (UV) CD spectra of AHATG3 peptide (75 μM) in the absence or presence of DOPC/DOPE (70/30) liposomes (6 mM). A peptide corresponding to the N-terminal ATG3 (1 to 22 amino acids) sequence and containing addi...
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AHATG3 cannot be substituted for by unrelated AHs in human ATG3 protein.(A) Far ultraviolet (UV) CD spectra of AHATG3 peptide (75 μM) in the absence or presence of DOPC/DOPE (70/30) liposomes (6 mM). A peptide corresponding to the N-terminal ATG3 (1 to 22 amino acids) sequence and containing additional C-terminal WK residues was used to facilitate titration by UV spectroscopy. The diameter of sonicated liposomes and liposomes extruded through 0.1- or 0.2-μm filters were 38, 100, and 147 nm, respectively. MRE, mean residue ellipticity. (B) Helicity at 222 nm as determined from the spectra shown in (A). Data represent the means ± SEM of three biological replicates. (C) An experimental scheme of ATG3 rescue assay using ATG3 chimeras. ATG3 KO MEFs expressing ATG3 WT, ATG3 ΔAH deletion mutant, or ATG3 chimera were used to analyze LC3 lipidation and LC3 puncta formation. The domain organization of ATG3-inGFP is shown: A GFP tag is inserted into the region after E125 residue. P21 and L23 in the conserved region following AHATG3 are coded in all ATG3 constructs. (D and F) LC3 flux assay. The indicated cells were starved for 6 hours with (B) or without 100 nM BafA1 (S) or cultured in full media (F). Cell lysates were analyzed by immunoblotting using the indicated antibodies. (E) LC3 puncta formation. The cells were starved for 1 hour, fixed, and stained with anti-LC3 antibody. The specimens were analyzed by an FV3000 confocal microscope. Arrowheads indicate the membrane localization of ATG3 chimeras. Scale bar, 10 μm. Differences were statistically analyzed by one-way analysis of variance (ANOVA) and Tukey multiple comparison test. ***P < 0.001.
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Collected and cropped from Sci Adv by CiteAb, provided under a CC-BY license
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In Sci Adv on 23 June 2023 by Nishimura, T., Lazzeri, G., et al.
Fig.3.F
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Low hydrophobicity and less bulky features are crucial for AHATG3 to be functional for LC3 lipidation.(A) Helical wheel representations of higher bulky-hydrophobic AHATG3 and less bulky-hydrophobic AHATG2A mutants. V8K mutation destroys the amphipathic character of AHATG3. Asterisks indicate the ...
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Low hydrophobicity and less bulky features are crucial for AHATG3 to be functional for LC3 lipidation.(A) Helical wheel representations of higher bulky-hydrophobic AHATG3 and less bulky-hydrophobic AHATG2A mutants. V8K mutation destroys the amphipathic character of AHATG3. Asterisks indicate the position of mutations. (B) LC3 flux assay of ATG3 KO cells expressing ATG3 WT or the indicated ATG3 mutants. The cells were starved for 6 hours with (B) or without 100 nM BafA1 (S) or cultured in full media (F). Cell lysates were analyzed by immunoblotting using the indicated antibodies. (C) Band intensity quantification of LC3-II. All data were normalized with those of HSP90. Data represent the means ± SEM of three biological replicates. (D) LC3 puncta formation. The cells were starved for 1 hour, fixed, and stained with anti-LC3 antibody. The specimens were analyzed by an FV3000 confocal microscope. Scale bar, 10 μm. (E) Quantification of the number of LC3 puncta. The thick and thin lines in the violin plot represent the medians and quartiles, respectively. The average number of LC3 puncta per cell was counted from randomly selected areas (n ≧ 14). (F) LC3 flux assay of ATG3 KO cells expressing ATG3 WT or the indicated ATG3 chimeras. Note that the ATG3 chimera carrying less bulky-hydrophobic mutant ATG2A-m1 showed the partial restoration of LC3 lipidation. (G) Band intensity quantification of LC3-II. All data were normalized with those of HSP90. Data represent the means ± SEM of three biological replicates. Differences were statistically analyzed by one-way ANOVA and Tukey multiple comparison test. ***P < 0.001.
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Collected and cropped from Sci Adv by CiteAb, provided under a CC-BY license
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In Front Cardiovasc Med on 22 November 2022 by Aranda, J. F., Pérez-García, A., et al.
Fig.6.B
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MiR-199a-5p suppresses ABCA1 expression. (A) Human and mouse ABCA1 3′UTRs sequences containing target sites for miR-199a/b-5p. Highlighted in bold are the sequences for miR-199a/b-5p-binding site and seed match. (B,C) Representative Western blot analysis of ABCA1 in mouse peritoneal macrophages t...
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MiR-199a-5p suppresses ABCA1 expression. (A) Human and mouse ABCA1 3′UTRs sequences containing target sites for miR-199a/b-5p. Highlighted in bold are the sequences for miR-199a/b-5p-binding site and seed match. (B,C) Representative Western blot analysis of ABCA1 in mouse peritoneal macrophages transfected with CM and miR-199a-5p or Ctrl-Inh or Inh-miR-199a-5p and stimulated with T090 3 μM for 12 h and Ac-LDL for 24 h. HSP90 was used as a loading control. Dashed lines separates different transfection conditions within the same Western blot. Data are expressed as relative expression levels and correspond to the means ± SEM from three independent experiments performed in triplicate. *P < 0.05, significantly different from CM or CI without treatment. (D) Luciferase reporter activity in COS-7 cells transfected with CM or miR-199a-5p mimic and the ABCA1 3’UTR [wild type (WT)] or the constructs containing the indicated point mutations (PM). Nucleotides highlighted in red indicate the point mutations in the miR-199a-5p-binding sites. Data are expressed as relative luciferase activity compared to the activity in control samples cotransfected with an equal concentration of CM and correspond to the means ± SEM of three experiments performed in triplicate. *P < 0.05, significantly different from cells cotransfected with CM and the WT or PM 3’UTR.
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Collected and cropped from Front Cardiovasc Med by CiteAb, provided under a CC-BY license
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In Front Cardiovasc Med on 22 November 2022 by Aranda, J. F., Pérez-García, A., et al.
Fig.7.B
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MiR-199 target site blockers and ABCA1 expression. (A) Schematic overview of LNA-enhanced target site blocker (TSB) mode of action. Without miR-199 TSB or with scramble TSB, miR-199/RISC complex binds to the 3’UTR of ABCA1 mRNA, thereby attenuating its expression. By contrast, addition of miR-199...
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MiR-199 target site blockers and ABCA1 expression. (A) Schematic overview of LNA-enhanced target site blocker (TSB) mode of action. Without miR-199 TSB or with scramble TSB, miR-199/RISC complex binds to the 3’UTR of ABCA1 mRNA, thereby attenuating its expression. By contrast, addition of miR-199 TSB blocks the binding of miR-199 and derepress ABCA1. (B) Representative Western blot of ABCA1 in mouse peritoneal macrophages transfected as indicated. transfected with synthetic miR-199a-5p mimic in the presence of scramble TSBs or specific miR-199 TSBs for 48 h. Lower panel shows relative ABCA1 protein expression normalized to HSP90 (n = 6). *P < 0.05, significantly different from cells cotransfected with CM and Ctrl TSB. (C) Luciferase reporter activity in COS-7 cells transfected with CM or miR-199a-5p mimic and the ABCA1 3’UTR in the presence of Ctrl TSB or miR-199a-5p TSB. Data are expressed as relative luciferase and correspond to the means ± SEM of three experiments performed in triplicate. **P < 0.01, significantly different from cells transfected with CM (left panel) or Ctrl TSB (right panel) ***P > 0.001. (D) Representation of the human 3’UTR region of ABCA1 where miR-199-5p binding site is located (red), and the overlapping with binding sites for the indicated miRNAs.
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Collected and cropped from Front Cardiovasc Med by CiteAb, provided under a CC-BY license
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In Cells on 16 August 2022 by Martín-Martín, Y., Pérez-García, A., et al.
Fig.5.B
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miR-31-5p regulates genes involved in insulin signaling pathway and controls IDE protein at the physiological level (a) DAVID analysis showing fractional difference analysis of the top enrichment pathways of miR-31-5p target genes. * p < 0.05; ** p < 0.01 (observed versus expected). (b) Represent...
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miR-31-5p regulates genes involved in insulin signaling pathway and controls IDE protein at the physiological level (a) DAVID analysis showing fractional difference analysis of the top enrichment pathways of miR-31-5p target genes. * p < 0.05; ** p < 0.01 (observed versus expected). (b) Representative Western blot analysis of IDE in SH and HepG2 cells transfected with control inhibitor (CI) or miR-31 inhibitor. HSP90 and Vinculin were used as loading controls.
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Collected and cropped from Cells by CiteAb, provided under a CC-BY license
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In Elife on 8 August 2022 by Yim, W. W., Yamamoto, H., et al.
Fig.1.A
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Processed ligand-bound HaloTag from HaloTag-LC3 is a quantifiable readout reflecting autophagic flux.(a) Immunoblotting of total cell lysates from wild-type and FIP200 knockout (KO) HeLa cells stably expressing monomeric GFP (mGFP)-LC3B or monomeric RFP (mRFP)-LC3B that was in nutrient-rich mediu...
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Processed ligand-bound HaloTag from HaloTag-LC3 is a quantifiable readout reflecting autophagic flux.(a) Immunoblotting of total cell lysates from wild-type and FIP200 knockout (KO) HeLa cells stably expressing monomeric GFP (mGFP)-LC3B or monomeric RFP (mRFP)-LC3B that was in nutrient-rich medium (0 hr) or incubated for 6 hr in starvation medium. (b) Immunoblotting and in-gel fluorescence detection of total cell lysates from wild-type and FIP200 KO HeLa cells stably expressing Halo-LC3B that was pulse-labeled for 20 min with 100 nM of tetramethylrhodamine (TMR)-conjugated ligand in nutrient-rich medium. The cells were immediately collected (0 hr) or incubated in starvation medium for 6 hr. (c) Same cells and labeling procedure as in (b) except that the cells were collected or incubated in starvation medium with or without 100 nM bafilomycin A1 (BafA) for 2 hr. (d) Same labeling procedure as in (b) except with HeLa cells stably expressing SNAP-tag (SNAP)-LC3B and TMR-conjugated ligand targeting SNAP. (e) Illustration of the fate of ligand-free and ligand-bound Halo-LC3B in autolysosomes. Halo-LC3B is processed by lysosomal hydrolases, releasing Halo from LC3B. When free from ligand, Halo is unstable and quickly degraded in lysosomes like LC3B. In contrast, ligand-bound Halo is stable against degradation and accumulates in lysosomes. Released Haloligand is detectable by immunoblot and, if a fluorescent ligand was used, in-gel fluorescence detection. The amount of free Haloligand separated from LC3B reflects the level of autophagic flux. (f) Immunoblotting and in-gel fluorescence detection of total cell lysates from wild-type HeLa cells stably expressing Halo-LC3B, pulse-labeled with 100 nM TMR-conjugated ligand in nutrient-rich medium for 20 min, and starved for the indicated durations. (g) Quantification of results shown in (f). HaloTMR band intensity was normalized by the sum of the band intensities HaloTMR-LC3B and HaloTMR. Mean values of data from three experiments are shown with red points that are traced by a line.Figure 1—source data 1.Uncropped blot images of Figure 1a, b, c, d and f, and Figure 1—figure supplement 1.Uncropped blot images of Figure 1a, b, c, d and f, and Figure 1—figure supplement 1.HaloTag-based processing assay can be conducted without a blocking agent.Wild-type HeLa cells stably expressing HaloTag (Halo)-LC3B, labeled with tetramethylrhodamine (TMR)-conjugated ligand or SF650-conjugated ligand at the indicated concentrations and in the indicated medium with or without SF650-conjugated ligand as a blocking agent. ‘Stop’ means that the samples were collected immediately after the pulse treatment.
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Collected and cropped from Elife by CiteAb, provided under a CC-BY license
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In Elife on 8 August 2022 by Yim, W. W., Yamamoto, H., et al.
Fig.4.A
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Bulk nonselective autophagic flux can be detected with HaloTag-GFP.(a,b) Immunoblotting and in-gel fluorescence detection of total cell lysates from wild-type, FIP200 knockout (KO), ATG3 KO, and ATG5 KO HeLa cells stably expressing HaloTag (Halo)-LC3B (a) or Halo-mGFP (a,b). After pulse-labeling ...
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Bulk nonselective autophagic flux can be detected with HaloTag-GFP.(a,b) Immunoblotting and in-gel fluorescence detection of total cell lysates from wild-type, FIP200 knockout (KO), ATG3 KO, and ATG5 KO HeLa cells stably expressing HaloTag (Halo)-LC3B (a) or Halo-mGFP (a,b). After pulse-labeling for 20 min with 100 nM tetramethylrhodamine (TMR)-conjugated ligand in nutrient-rich medium, the cells were collected or incubated for 6 hr (a) or the indicated times (b) in starvation medium. (c) Quantification of time course results shown in (b). Mean values of three experiments are shown with red points that are traced with a line. (d) Fluorescence images of wild-type and FIP200 KO HeLa cells stably expressing Halo-mGFP in nutrient-rich medium or after 6 hr of starvation. The media contained 200 nM of SF650-conjugated ligand. Scale bar = 10 µm (main), 2 µm (magnified images). (e) Quantification of the area of HaloSF650 signal that exceeded a 98th percentile intensity threshold in cells described in (d) at the indicated durations of starvation. n=42–48 cells. In box plots, solid bars indicate medians, boxes indicate the interquartile range (25th–75th percentile), and whiskers indicate the largest and smallest values within 1.5 times the interquartile range.Figure 4—source data 1.Uncropped blot images of Figure 4a and b.Uncropped blot images of Figure 4a and b.Bulk nonselective autophagic flux can be detected with Halo-GFP.(a) Fluorescence images of wild-type HeLa cells stably expressing lysosomal marker LAMP1-mRuby3 and HaloTag (Halo)-mGFP under nutrient-rich conditions or after 6 hr under starvation conditions in the presence of 200 nM SF650-conjugated ligand. (b) Time-lapse montage of wild-type HeLa cells stably expressing Halo-mGFP cells in starvation medium containing 200 nM SF650-conjugated ligand and 75 nM LysoTracker Red. Images were taken after the indicated durations of starvation. Scale bar = 10 µm (main), 2 µm (inset, inset montage) (a, b).
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Collected and cropped from Elife by CiteAb, provided under a CC-BY license
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In Cell Death Discov on 25 January 2022 by Date, Y., Matsuura, A., et al.
Fig.2.K
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Cytochalasin D treatment induces lysosomal degradation of TRiC.A, B Analysis of lysosomal degradation of TRiC by flow cytometry. HeLa TCP1α-RG cells were treated with tunicamycin, CCCP, cytochalasin D, nocodazole, or arsenic for 24 h and analyzed by flow cytometry. Dot plots show the lysosomal TR...
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Cytochalasin D treatment induces lysosomal degradation of TRiC.A, B Analysis of lysosomal degradation of TRiC by flow cytometry. HeLa TCP1α-RG cells were treated with tunicamycin, CCCP, cytochalasin D, nocodazole, or arsenic for 24 h and analyzed by flow cytometry. Dot plots show the lysosomal TRiC gate-positive cell population (%). The bar graph shows the TRiC degradation ratio: the compound-treated lysosomal TRiC gate-positive cell population (%)/DMSO-treated lysosomal TRiC gate-positive cell population (%) (n = 3). The data are presented as the means ± SEM. *P < 0.05, t-test. C, D Time- and concentration-dependent lysosomal TRiC degradation by cytochalasin D. HeLa TCP1α-RG cells were treated with DMSO or cytochalasin D (C: 10 µM for 10 or 24 h, C) (D: 1 or 10 μM for 24 h) and analyzed by flow cytometry. The histogram shows the number of cells on the vertical axis and the RFP/GFP ratio on the horizontal axis. E TRiC-RG is transported to lysosomes. HeLa TCP1α-RG cells were treated with DMSO or cytochalasin D for 24 h, fixed and stained with anti-lamp1 antibody, and then analyzed by confocal microscopy. Scale bar, 20 µm. F The box plot shows the RFP/Lamp1 colocalization in HeLa TCP1α-RG cells treated with DMSO or cytochalasin D (Cyto D) for 24 h (n = 30 cells). **** P < 0.0001, t-test. G–I Lysosomal TRiC degradation is suppressed by bafilomycin A1 treatment. HeLa TCP1α-RG cells were treated with DMSO or cytochalasin D with or without bafilomycin A1 (baf) for 24 h and analyzed by flow cytometry. Dot plots show the lysosomal TRiC gate-positive cell population (%). The bar graph shows the TRiC degradation ratio of H (n = 3). The data are presented as the means ± SEM. *P < 0.05, ANOVA Tukey’s test. J Analysis of lysosomal TRiC degradation by TCP1α-RG cleavage assay. HeLa TCP1α-RG cells were treated with DMSO or cytochalasin D, with or without bafilomycin A1 (baf) for 24 h, and analyzed by immunoblotting using antibodies against RFP and HSP90. K Suppression of actin dynamics causes lysosomal TRiC degradation. HeLa TCP1α-RFP-GFP cells were treated with DMSO, cytochalasin D (Cyto D), latrunculin A (Lat A) or cucurbitacin E (Cucu E) with or without bafilomycin A1 (baf) for 24 h and analyzed by immunoblotting using antibodies against RFP and HSP90.
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Collected and cropped from Cell Death Discov by CiteAb, provided under a CC-BY license
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In Cell Death Discov on 25 January 2022 by Date, Y., Matsuura, A., et al.
Fig.2.J
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Cytochalasin D treatment induces lysosomal degradation of TRiC.A, B Analysis of lysosomal degradation of TRiC by flow cytometry. HeLa TCP1α-RG cells were treated with tunicamycin, CCCP, cytochalasin D, nocodazole, or arsenic for 24 h and analyzed by flow cytometry. Dot plots show the lysosomal TR...
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Cytochalasin D treatment induces lysosomal degradation of TRiC.A, B Analysis of lysosomal degradation of TRiC by flow cytometry. HeLa TCP1α-RG cells were treated with tunicamycin, CCCP, cytochalasin D, nocodazole, or arsenic for 24 h and analyzed by flow cytometry. Dot plots show the lysosomal TRiC gate-positive cell population (%). The bar graph shows the TRiC degradation ratio: the compound-treated lysosomal TRiC gate-positive cell population (%)/DMSO-treated lysosomal TRiC gate-positive cell population (%) (n = 3). The data are presented as the means ± SEM. *P < 0.05, t-test. C, D Time- and concentration-dependent lysosomal TRiC degradation by cytochalasin D. HeLa TCP1α-RG cells were treated with DMSO or cytochalasin D (C: 10 µM for 10 or 24 h, C) (D: 1 or 10 μM for 24 h) and analyzed by flow cytometry. The histogram shows the number of cells on the vertical axis and the RFP/GFP ratio on the horizontal axis. E TRiC-RG is transported to lysosomes. HeLa TCP1α-RG cells were treated with DMSO or cytochalasin D for 24 h, fixed and stained with anti-lamp1 antibody, and then analyzed by confocal microscopy. Scale bar, 20 µm. F The box plot shows the RFP/Lamp1 colocalization in HeLa TCP1α-RG cells treated with DMSO or cytochalasin D (Cyto D) for 24 h (n = 30 cells). **** P < 0.0001, t-test. G–I Lysosomal TRiC degradation is suppressed by bafilomycin A1 treatment. HeLa TCP1α-RG cells were treated with DMSO or cytochalasin D with or without bafilomycin A1 (baf) for 24 h and analyzed by flow cytometry. Dot plots show the lysosomal TRiC gate-positive cell population (%). The bar graph shows the TRiC degradation ratio of H (n = 3). The data are presented as the means ± SEM. *P < 0.05, ANOVA Tukey’s test. J Analysis of lysosomal TRiC degradation by TCP1α-RG cleavage assay. HeLa TCP1α-RG cells were treated with DMSO or cytochalasin D, with or without bafilomycin A1 (baf) for 24 h, and analyzed by immunoblotting using antibodies against RFP and HSP90. K Suppression of actin dynamics causes lysosomal TRiC degradation. HeLa TCP1α-RFP-GFP cells were treated with DMSO, cytochalasin D (Cyto D), latrunculin A (Lat A) or cucurbitacin E (Cucu E) with or without bafilomycin A1 (baf) for 24 h and analyzed by immunoblotting using antibodies against RFP and HSP90.
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Collected and cropped from Cell Death Discov by CiteAb, provided under a CC-BY license
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In Nat Commun on 17 December 2021 by Yao, F., Deng, Y., et al.
Fig.3.H
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LIFR negatively regulates NF-κB signaling and LCN2 in the liver.a Volcano plot of genes upregulated (red) or downregulated (blue) in Lifrfl/fl;Alb-Cre mice (n = 3) relative to Lifrfl/fl mice (n = 2). Statistical analysis of RNA-seq data was performed using Cuffdiff and P values are false discover...
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LIFR negatively regulates NF-κB signaling and LCN2 in the liver.a Volcano plot of genes upregulated (red) or downregulated (blue) in Lifrfl/fl;Alb-Cre mice (n = 3) relative to Lifrfl/fl mice (n = 2). Statistical analysis of RNA-seq data was performed using Cuffdiff and P values are false discovery rate (FDR)-adjusted. b Cytokine arrays of the conditioned medium of Lifr-knockout PHM cells. Boxed: the top five upregulated (red) and downregulated (blue) cytokines. c qPCR of Lcn2 in Lifr-knockout PHM cells. n = 3 samples. d ELISA of lipocalin 2 in the conditioned medium of Lifr-knockout PHM cells. n = 5, 4, and 5 wells from left to right. e ELISA of lipocalin 2 in the serum of 3-month-old Lifrfl/fl and Lifrfl/fl;Alb-Cre mice. n = 4 mice. f Pathway analysis of Lifr-knockout PHM cells with or without LIFR add-back. g, h Immunoblotting of p-p65, p65, and LIFR in LIFR-knockdown (g) and LIFR-overexpressing (h) Mahlavu cells. i, j Pathway analysis of livers of Lifrfl/fl and Lifrfl/fl;Alb-Cre mice that received hydrodynamic injection of plasmids expressing the Sleeping Beauty transposase and oncogenes (i: β-catenin + YAP; j: myrAKT + RasV12). k, l qPCR of Lcn2 in livers described in i and j, respectively. n = 3 samples per mouse; n = 2 mice per group. m, n Immunohistochemical staining (m) and quantification (n) of Lcn2 in livers described in i and j, respectively. n = 6 mice. o Immunoblotting of Lifr, p-p65, p65, p-Stat3, Stat3, and Gapdh in livers of Lifrfl/fl and Lifrfl/fl;Cre-ERT2 mice, 28 days after hydrodynamic injection of plasmids expressing the Sleeping Beauty transposase, myrAKT, and RasV12. From day 7, all mice received 5-day tamoxifen treatment. p, q Immunohistochemical staining (p) and quantification (q) of Lcn2 in livers of the mice that received control or LIFR-expressing adenovirus 3 days and 17 days after hydrodynamic injection of plasmids expressing the Sleeping Beauty transposase, myrAKT, and RasV12. Scale bars, 200 μm. n = 4 mice. Statistical significance in c–e, n, and q was determined by a two-tailed unpaired t-test. Error bars are s.e.m. Source data are provided as a Source Data file.
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Collected and cropped from Nat Commun by CiteAb, provided under a CC-BY license
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In Front Cell Dev Biol on 26 November 2021 by Fu, X. W. & Song, C. Q.
Fig.11.R
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Sensitivity correlation analysis between the independent prognostic genes and drugs based on the CellMiner Database. (A–P) Correlation analysis. (Q) Relative mRNA expression of GSDME following treatment with Lenvatinib for 48 h in Huh7. (R) Analysis of GSDME and GSDME-N terminal in Huh7 treated w...
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Sensitivity correlation analysis between the independent prognostic genes and drugs based on the CellMiner Database. (A–P) Correlation analysis. (Q) Relative mRNA expression of GSDME following treatment with Lenvatinib for 48 h in Huh7. (R) Analysis of GSDME and GSDME-N terminal in Huh7 treated with Lenvatinib for 48 h by western blotting. *p < 0.05, **p < 0.01, ***p < 0.001.
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Collected and cropped from Front Cell Dev Biol by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 30 August 2021 by Luo, H., Zhou, Z., et al.
Fig.7.A
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CHFR promotes apoptosis and correlates with good clinical outcome in malignant breast cancer.a LM2 cells were treated with or without DOX and TSA as indicated and immunoblotted with the FASN, Cleaved Caspase-3, and HSP90 antibodies. b, c SUM159 cells were treated as described in a and stained wit...
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CHFR promotes apoptosis and correlates with good clinical outcome in malignant breast cancer.a LM2 cells were treated with or without DOX and TSA as indicated and immunoblotted with the FASN, Cleaved Caspase-3, and HSP90 antibodies. b, c SUM159 cells were treated as described in a and stained with propidium iodide and Annexin V-FITC and then analyzed by flow cytometry (b); c showed the percentage of early apoptotic (Annexin V+PI−; c) cells in SUM159 cells. d Tumor growth curves of nude mice with subcutaneous injection of LM2 cells. From day 7, mice received every 3 days intraperitoneal injections of doxorubicin with or without TSA. n = 3 mice per group. e Kaplan–Meier curves showing the distant relapse-free survival of patients with high or low expression of CHFR in the mesenchymal/basal subtype breast tumors. f Kaplan–Meier curves showing the distant relapse-free survival of patients with high or low expression of CHFR in the basal-like 1 subtype breast tumors. Significance of Mock versus DOX or TSA is shown. n = 3 wells per group. Data in c, d are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t test. The experiments were repeated three times. Statistical significance in e, f was determined by log-rank test.
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Int J Mol Sci on 17 November 2020 by Benito-Vicente, A., Uribe, K. B., et al.
Fig.2.E
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miR-27b modulation affects insulin signaling in Huh7 cell line: (A,B) miR-27b levels in Huh7 cells transfected with (A) 40 nM control mimic and mimic miR-27b or (B) 40 nM control inhibitor and inhibitor miR-27b; (C,D) mRNA expression of ABCA1, INSR, IRS1, AKT2, FOXO1 and GSK3β in Huh7 cells trans...
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miR-27b modulation affects insulin signaling in Huh7 cell line: (A,B) miR-27b levels in Huh7 cells transfected with (A) 40 nM control mimic and mimic miR-27b or (B) 40 nM control inhibitor and inhibitor miR-27b; (C,D) mRNA expression of ABCA1, INSR, IRS1, AKT2, FOXO1 and GSK3β in Huh7 cells transfected with (C) 40 nM control mimic and mimic miR-27b or (D) 40 nM control inhibitor and inhibitor miR-27b; (E) Western blot analysis of ABCA1, INSR, IRS1, AKT2, FOXO1 and GSK3β expression in Huh7 cell transfected with miR-27b mimic or miR-27b inhibitor and their respective controls; (F) Western blot analysis of ABCA1, INSR, IRS1, AKT2, FOXO1 and GSK3β expression in Huh7 cell transfected with miR-27b mimic or miR-27b inhibitor and their respective controls; and (G) analysis of miR-27b targeting on the 3′UTR of human INSR and IRS1 by luciferase reporter assay. * p < 0.05 (significantly different from cells transfected with the CM or CI). Data are presented as the mean ± SEM. n = 3 independent experiments ((A–D,G and right-handed panels of (E,F)). Data correspond to results from a representative experiment among three that gave similar results in left-handed panels of (E,F).
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
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In Int J Mol Sci on 17 November 2020 by Benito-Vicente, A., Uribe, K. B., et al.
Fig.4.G
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miR-27b modulation affects insulin signaling in wild type mice: (A) schematic representation of mice treatment; (B,C) miR-27b levels in wild type mice treated with (B) null AAV or AAV miR-27b or (C) LNA control or LNA miR-27b inhibitor; (D,E) mRNA expression of Abca1, Insr, Irs1, Akt2, Foxo1 and ...
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miR-27b modulation affects insulin signaling in wild type mice: (A) schematic representation of mice treatment; (B,C) miR-27b levels in wild type mice treated with (B) null AAV or AAV miR-27b or (C) LNA control or LNA miR-27b inhibitor; (D,E) mRNA expression of Abca1, Insr, Irs1, Akt2, Foxo1 and Gsk3β in wild type mice treated with (D) null AAV or AAV miR-27b or (E) LNA control or LNA miR-27b inhibitor; (F) Western blot analysis of ABCA1, INSR, IRS1, AKT2, FOXO1 and GSK3β expression in wild-type mice treated either with AAV-miR-27b or AAV control; (G) Western blot analysis of ABCA1, INSR, IRS1, AKT2, FOXO1 and GSK3β expression in wild-type mice treated either with LNA-miR-27b inhibitor or LNA control; and (H) Western blot analysis of AKT2 phosphorylation in wild type mice treated with LNA miR-27b inhibitor and its respective control. * p < 0.05 (significantly different from cells transfected with the CM or CI). Data are presented as the mean ± SEM. n = 3 independent experiments ((B–E) and right-handed panels of (F–H)). Western blot images correspond to results from a representative experiment among three that gave similar results in (D,G).
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Collected and cropped from Int J Mol Sci by CiteAb, provided under a CC-BY license
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In Biol Res on 25 September 2020 by Yang, J., Lee, S. J., et al.
Fig.3.A
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MATR3 promotes apoptotic cell death gauged by PARP cleavage. MDA-MB-231 (a introduced with empty vector or MATR3-overexpression vector) and BT549 cells (b introduced with non-silencing control or sh-MATR3) were treated with 0.5% DMSO (−) or 0.5 μM Staurosporine (+) for 24 h and subjected to weste...
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MATR3 promotes apoptotic cell death gauged by PARP cleavage. MDA-MB-231 (a introduced with empty vector or MATR3-overexpression vector) and BT549 cells (b introduced with non-silencing control or sh-MATR3) were treated with 0.5% DMSO (−) or 0.5 μM Staurosporine (+) for 24 h and subjected to western blotting. Cleaved PARP (cPARP) was detected with a specific antibody. The lower panels demonstrate the quantitation of the relative normalized cPARP protein levels
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Collected and cropped from Biol Res by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 4 May 2020 by Wu, Y. H., Chou, T. F., et al.
Fig.5.C
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Sensitivity to TNF-induced septic shock in Dapk1−/− mice and DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a, b Control and Dapk1−/− mice (n = 4 in each group) were injected with mouse TNF (1.0 μg/g body weight), and survival (a) and rectal body temperatu...
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Sensitivity to TNF-induced septic shock in Dapk1−/− mice and DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a, b Control and Dapk1−/− mice (n = 4 in each group) were injected with mouse TNF (1.0 μg/g body weight), and survival (a) and rectal body temperature (b) were determined at the indicated time points. P values (a) for Long-rank (Mantel-–Cox) test. Mean ± SD (b) are shown. *P < 0.05, **P < 0.01 (b) for two-way ANOVA followed by a Tukey’s multiple comparison test. (c) Normal TNF-triggered activation or NF-κB, ERK, JNK, and AKT in Dapk1−/− BMDMs. WT and Dapk1−/− BMDMs were treated with TNF (20 ng/ml), and cell lysates were prepared and levels of pIκBα, IκBα, pERK, ERK, pJNK, JNK, pAKT, and AKT were determined for the indicated time points. (d, f) Diminished TNF-induced phosphorylation of RIPK1(S321), MK2 and p38 MAPK in Dapk1−/− BMDMs. WT and Dapk1−/− BMDMs were treated with TNF (20 ng/ml), and levels of pIKK, IKK, pRIPK1(S321), RIPK1, p-p38, p38 (d), pMK2 and MK2 (f) were determined in cell lysates for the indicated time points. Data are representative of three independent experiments. (e) The extents of RIPK1(S321) and p38 MAPK activation from three independent experiments in (d) were quantitated using normalized intensity of pRIPK1(S321) and p-p38 in WT BMDMs at 15 min as 1. Mean ± SD are shown. *P < 0.05, ***P < 0.001 for two-way ANOVA followed by a Sidak’s multiple comparison test. (g) Inhibition of p38 MAPK or MK2 confers susceptibility to necroptosis in WT macrophages. WT BMDMs were treated with zVAD, AT-406, SB203589 (1 μM) and PF3644022 (2 μM), as indicated, and the extent of necroptosis determined. Values are mean ± SD of triplicates in a single experiment. **p < 0.01, ***p < 0.001 for unpaired t-test. Results have been repeated in two independent experiments.
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 4 May 2020 by Wu, Y. H., Chou, T. F., et al.
Fig.5.D
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Sensitivity to TNF-induced septic shock in Dapk1−/− mice and DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a, b Control and Dapk1−/− mice (n = 4 in each group) were injected with mouse TNF (1.0 μg/g body weight), and survival (a) and rectal body temperatu...
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Sensitivity to TNF-induced septic shock in Dapk1−/− mice and DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a, b Control and Dapk1−/− mice (n = 4 in each group) were injected with mouse TNF (1.0 μg/g body weight), and survival (a) and rectal body temperature (b) were determined at the indicated time points. P values (a) for Long-rank (Mantel-–Cox) test. Mean ± SD (b) are shown. *P < 0.05, **P < 0.01 (b) for two-way ANOVA followed by a Tukey’s multiple comparison test. (c) Normal TNF-triggered activation or NF-κB, ERK, JNK, and AKT in Dapk1−/− BMDMs. WT and Dapk1−/− BMDMs were treated with TNF (20 ng/ml), and cell lysates were prepared and levels of pIκBα, IκBα, pERK, ERK, pJNK, JNK, pAKT, and AKT were determined for the indicated time points. (d, f) Diminished TNF-induced phosphorylation of RIPK1(S321), MK2 and p38 MAPK in Dapk1−/− BMDMs. WT and Dapk1−/− BMDMs were treated with TNF (20 ng/ml), and levels of pIKK, IKK, pRIPK1(S321), RIPK1, p-p38, p38 (d), pMK2 and MK2 (f) were determined in cell lysates for the indicated time points. Data are representative of three independent experiments. (e) The extents of RIPK1(S321) and p38 MAPK activation from three independent experiments in (d) were quantitated using normalized intensity of pRIPK1(S321) and p-p38 in WT BMDMs at 15 min as 1. Mean ± SD are shown. *P < 0.05, ***P < 0.001 for two-way ANOVA followed by a Sidak’s multiple comparison test. (g) Inhibition of p38 MAPK or MK2 confers susceptibility to necroptosis in WT macrophages. WT BMDMs were treated with zVAD, AT-406, SB203589 (1 μM) and PF3644022 (2 μM), as indicated, and the extent of necroptosis determined. Values are mean ± SD of triplicates in a single experiment. **p < 0.01, ***p < 0.001 for unpaired t-test. Results have been repeated in two independent experiments.
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 4 May 2020 by Wu, Y. H., Chou, T. F., et al.
Fig.5.F
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Sensitivity to TNF-induced septic shock in Dapk1−/− mice and DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a, b Control and Dapk1−/− mice (n = 4 in each group) were injected with mouse TNF (1.0 μg/g body weight), and survival (a) and rectal body temperatu...
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Sensitivity to TNF-induced septic shock in Dapk1−/− mice and DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a, b Control and Dapk1−/− mice (n = 4 in each group) were injected with mouse TNF (1.0 μg/g body weight), and survival (a) and rectal body temperature (b) were determined at the indicated time points. P values (a) for Long-rank (Mantel-–Cox) test. Mean ± SD (b) are shown. *P < 0.05, **P < 0.01 (b) for two-way ANOVA followed by a Tukey’s multiple comparison test. (c) Normal TNF-triggered activation or NF-κB, ERK, JNK, and AKT in Dapk1−/− BMDMs. WT and Dapk1−/− BMDMs were treated with TNF (20 ng/ml), and cell lysates were prepared and levels of pIκBα, IκBα, pERK, ERK, pJNK, JNK, pAKT, and AKT were determined for the indicated time points. (d, f) Diminished TNF-induced phosphorylation of RIPK1(S321), MK2 and p38 MAPK in Dapk1−/− BMDMs. WT and Dapk1−/− BMDMs were treated with TNF (20 ng/ml), and levels of pIKK, IKK, pRIPK1(S321), RIPK1, p-p38, p38 (d), pMK2 and MK2 (f) were determined in cell lysates for the indicated time points. Data are representative of three independent experiments. (e) The extents of RIPK1(S321) and p38 MAPK activation from three independent experiments in (d) were quantitated using normalized intensity of pRIPK1(S321) and p-p38 in WT BMDMs at 15 min as 1. Mean ± SD are shown. *P < 0.05, ***P < 0.001 for two-way ANOVA followed by a Sidak’s multiple comparison test. (g) Inhibition of p38 MAPK or MK2 confers susceptibility to necroptosis in WT macrophages. WT BMDMs were treated with zVAD, AT-406, SB203589 (1 μM) and PF3644022 (2 μM), as indicated, and the extent of necroptosis determined. Values are mean ± SD of triplicates in a single experiment. **p < 0.01, ***p < 0.001 for unpaired t-test. Results have been repeated in two independent experiments.
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 4 May 2020 by Wu, Y. H., Chou, T. F., et al.
Fig.6.E
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DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a DAPK1-deficiency does not affect MKK3 activation. WT and Dapk1−/− BMDMs were treated with TNF, and levels of pMKK3 and MKK3 were determined in cell lysates. b DAPK1[K42A] increases resistance to necroptosis ...
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DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a DAPK1-deficiency does not affect MKK3 activation. WT and Dapk1−/− BMDMs were treated with TNF, and levels of pMKK3 and MKK3 were determined in cell lysates. b DAPK1[K42A] increases resistance to necroptosis in DAPK1-deficient cells. DAPK1−/− HT-29 cells were transfected with empty vector (EV), or transfected with WT DAPK1 or DAPK1[K42A]. Cells were treated with zVAD (Ctrl) or zVAD + BV6, and viability determined by ATP assay. c Either WT DAPK or DAPK1[K42A] increases p38 MAPK activation in DAPK1-null cells. DAPK1−/− HT-29 cells, mock transduced, transduced with WT DAPK1 or DAPK1[K42A] were treated with TNF (20 ng/ml) for the indicated time points, and the levels of DAPK1, p38 MAPK and phospho-p38 MAPK were determined. d Overexpression of p38 MAPK inhibits necroptosis. DAPK1−/− HT-29 cells were transfected with empty vector (EV), p38 MAPK or MKK3. The extent of necroptosis induced by zVAD+BV6 was determined by ATP release. e Interaction between DAPK1 and p38 MAPK. HEK293T cells were transfected with DAPK1-FLAG and/or p38-HA as indicated. Cell lysates were prepared 24 later, and precipitated with anti-FLAG or anti-HA. f p38 MAPK binds DAPK in vitro. Recombinant human p38 MAPK (200 ng) was incubated with purified DAPK (100 ng), as indicated. p38 MAPK was pulled down by anti-p38 MAPK (ab170099, Abcam), and the presence of DAPK and p38 MAPK in immunoprecipitants determined by anti-FLAG and anti-His, respectively. Results have been independently confirmed using another anti-p38 MAPK (7218, Cell Signaling). g DAPK1 promotes MKK3-directed p38 MAPK phosphorylation. Recombinant p38 MAPK (50 ng), MKK3 (100 ng), and DAPK1-FLAG (25 ng or 75 ng) was incubated as indicated in ATP-containing kinase buffer at 30 °C for 1 h. The amounts of p38 MAPK, MKK3, DAPK1 and phospho-p38 MAPK was determined by immunoblots. h Binding of different DAPK1 mutants to p38 MAPK. HEK293T cells were transfected with different mutants of DAPK1-FLAG and p38-HA as indicated. The presence of p38-HA in anti-FLAG precipitates was determined. i Failure of DAPK1(∆Cyt) to increase p38 MAPK activation. DAPK1, DAPK1(∆Cyt) or DAPK1(∆CAM) was evaluated for its ability to increase MKK3-directed p38 MAPK activation described in (g). Values are mean ± SD of triplicates in a single experiment. *P < 0.05, ***P < 0.001 (b, d) for two-way ANOVA followed by a Tukey’s multiple comparison test. Results have been repeated in three (a–c, e, f) or two (d, g–i) independent experiments.
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
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In Cell Death Dis on 4 May 2020 by Wu, Y. H., Chou, T. F., et al.
Fig.6.A
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WB
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DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a DAPK1-deficiency does not affect MKK3 activation. WT and Dapk1−/− BMDMs were treated with TNF, and levels of pMKK3 and MKK3 were determined in cell lysates. b DAPK1[K42A] increases resistance to necroptosis ...
more
DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a DAPK1-deficiency does not affect MKK3 activation. WT and Dapk1−/− BMDMs were treated with TNF, and levels of pMKK3 and MKK3 were determined in cell lysates. b DAPK1[K42A] increases resistance to necroptosis in DAPK1-deficient cells. DAPK1−/− HT-29 cells were transfected with empty vector (EV), or transfected with WT DAPK1 or DAPK1[K42A]. Cells were treated with zVAD (Ctrl) or zVAD + BV6, and viability determined by ATP assay. c Either WT DAPK or DAPK1[K42A] increases p38 MAPK activation in DAPK1-null cells. DAPK1−/− HT-29 cells, mock transduced, transduced with WT DAPK1 or DAPK1[K42A] were treated with TNF (20 ng/ml) for the indicated time points, and the levels of DAPK1, p38 MAPK and phospho-p38 MAPK were determined. d Overexpression of p38 MAPK inhibits necroptosis. DAPK1−/− HT-29 cells were transfected with empty vector (EV), p38 MAPK or MKK3. The extent of necroptosis induced by zVAD+BV6 was determined by ATP release. e Interaction between DAPK1 and p38 MAPK. HEK293T cells were transfected with DAPK1-FLAG and/or p38-HA as indicated. Cell lysates were prepared 24 later, and precipitated with anti-FLAG or anti-HA. f p38 MAPK binds DAPK in vitro. Recombinant human p38 MAPK (200 ng) was incubated with purified DAPK (100 ng), as indicated. p38 MAPK was pulled down by anti-p38 MAPK (ab170099, Abcam), and the presence of DAPK and p38 MAPK in immunoprecipitants determined by anti-FLAG and anti-His, respectively. Results have been independently confirmed using another anti-p38 MAPK (7218, Cell Signaling). g DAPK1 promotes MKK3-directed p38 MAPK phosphorylation. Recombinant p38 MAPK (50 ng), MKK3 (100 ng), and DAPK1-FLAG (25 ng or 75 ng) was incubated as indicated in ATP-containing kinase buffer at 30 °C for 1 h. The amounts of p38 MAPK, MKK3, DAPK1 and phospho-p38 MAPK was determined by immunoblots. h Binding of different DAPK1 mutants to p38 MAPK. HEK293T cells were transfected with different mutants of DAPK1-FLAG and p38-HA as indicated. The presence of p38-HA in anti-FLAG precipitates was determined. i Failure of DAPK1(∆Cyt) to increase p38 MAPK activation. DAPK1, DAPK1(∆Cyt) or DAPK1(∆CAM) was evaluated for its ability to increase MKK3-directed p38 MAPK activation described in (g). Values are mean ± SD of triplicates in a single experiment. *P < 0.05, ***P < 0.001 (b, d) for two-way ANOVA followed by a Tukey’s multiple comparison test. Results have been repeated in three (a–c, e, f) or two (d, g–i) independent experiments.
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Collected and cropped from Cell Death Dis by CiteAb, provided under a CC-BY license
Image 1 of 62