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Fig.4.E showing Western Blotting from the publication: Chaperone-mediated autophagy modulates Snail protein stability: implications for breast cancer metastasis.

Fig.2.A showing Western Blotting from the publication: NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension.

Fig.6.C showing Western Blotting from the publication: CD109 Is a Critical Determinant of EGFR Expression and Signaling, and Tumorigenicity in Squamous Cell Carcinoma Cells.

Fig.6.B showing Western Blotting from the publication: CD109 Is a Critical Determinant of EGFR Expression and Signaling, and Tumorigenicity in Squamous Cell Carcinoma Cells.

Fig.2.A showing Immunocytochemistry-immunofluorescence from the publication: Reversion-inducing cysteine-rich protein with Kazal motifs and MT1-MMP promote the formation of robust fibrillin fibers.

Fig.6.A showing Immunocytochemistry-immunofluorescence from the publication: Reversion-inducing cysteine-rich protein with Kazal motifs and MT1-MMP promote the formation of robust fibrillin fibers.

Fig.6.C showing Western Blotting from the publication: Transglutaminase-2 facilitates extracellular vesicle-mediated establishment of the metastatic niche.

Fig.6.B showing Western Blotting from the publication: CD109 acts as a gatekeeper of the epithelial trait by suppressing epithelial to mesenchymal transition in squamous cell carcinoma cells in vitro.

Fig.4.B showing Western Blotting from the publication: The RECK tumor-suppressor protein binds and stabilizes ADAMTS10.

Fig.4.F showing Western Blotting from the publication: The RECK tumor-suppressor protein binds and stabilizes ADAMTS10.

Fig.3.C showing Western Blotting from the publication: Ougan (Citrus reticulata cv. Suavissima) flavedo extract suppresses cancer motility by interfering with epithelial-to-mesenchymal transition in SKOV3 cells.

11 images found

Metabolic adaptations of micrometastases alter EV production to generate invasive microenvironments.

In The Journal of Cell Biology on 4 August 2025 by Gounis, M., Campos, A., et al.

Altered cellular metabolism has been associated with the acquisition of invasive phenotypes during metastasis. To study this, we combined a genetically engineered mouse model of mammary carcinoma with syngeneic transplantation and primary tumor resection to generate isogenic cells from primary tumors and their corresponding lung micrometastases. Metabolic analyses indicated that micrometastatic cells increase proline production at the expense of glutathione synthesis, leading to a reduction in total glutathione levels. Micrometastatic cells also have altered sphingomyelin metabolism, leading to increased intracellular levels of specific ceramides. The combination of these metabolic adaptations alters extracellular vesicle (EV) production to render the microenvironment more permissive for invasion. Indeed, micrometastatic cells shut down Rab27-dependent production of EVs and, instead, switch on neutral sphingomyelinase-2 (nSM2)-dependent EV release. EVs released in an nSM2-dependent manner from micrometastatic cells, in turn, influence the ability of fibroblasts to deposit extracellular matrix, which promotes cancer cell invasiveness. These data provide evidence that metabolic rewiring drives invasive processes in metastasis by influencing EV release.
© 2025 Gounis et al.

Phospholipase D6 activates Wnt/β-catenin signaling through mitochondrial metabolic reprogramming to promote tumorigenesis in colorectal cancer.

In Experimental & Molecular Medicine on 1 April 2025 by Lee, H. J., Lim, S. H., et al.

Phospholipase D6 (PLD6) is a critical enzyme involved in mitochondrial fusion with a key role in spermatogenesis. However, the role of PLD6 in cancer remains unknown. Notably, Wnt signaling, energy metabolism and mitochondrial function show complex interactions in colorectal cancer (CRC) progression. Here we found that PLD6 is highly expressed in CRC and positively correlated with poor prognosis. We present a novel function of PLD6 in activating Wnt/β-catenin signaling by enhancing mitochondrial metabolism. PLD6 depletion suppresses the oncogenic properties of CRC cells and impairs mitochondrial respiration, leading to reduced mitochondrial length, membrane potential, calcium levels and reactive oxygen species. PLD6 depletion also disrupts mitochondrial metabolic reprogramming by inhibiting the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation, resulting in altered intracellular levels of citrate and acetyl-CoA-both key modulators of Wnt/β-catenin activation. PLD6-mediated acetyl-CoA production enhances β-catenin stability by promoting its acetylation via the acetyltransferases CREB-binding protein and P300/CREB-binding-protein-associated factor. Consequently, PLD6 ablation reduces cancer stem cell-associated gene expression downstream of Wnt/β-catenin signaling, suppressing stem-like traits and chemoresistance to 5-fluorouracil. Furthermore, PLD6 depletion attenuates CRC tumorigenesis in both subcutaneous and orthotopic tumor models. Overall, PLD6 acts as an oncogenic switch by promoting mitochondria-mediated retrograde signaling, thereby regulating Wnt signaling in CRC.
© 2025. The Author(s).

MSK1 promotes colorectal cancer metastasis by increasing Snail protein stability through USP5-mediated Snail deubiquitination.

In Experimental & Molecular Medicine on 1 April 2025 by Hong, K. S., Ryu, K. J., et al.

Mitogen- and stress-activated protein kinase 1 (MSK1), a Ser/Thr kinase, phosphorylates nuclear proteins to increase their stability and DNA-binding affinity. Despite the role of MSK1 in promoting cancer progression in colorectal cancer (CRC), the precise molecular mechanisms remain unelucidated. Here we show that MSK1 expression induces the epithelial-mesenchymal transition (EMT) process and increases CRC cell metastasis. Furthermore, we discovered that MSK1 interacts with Snail, a key EMT regulator, and increases its stability by inhibiting ubiquitin-mediated proteasomal degradation. Importantly, MSK1 increased Snail protein stability by promoting deubiquitination rather than inhibiting its ubiquitination. Finally, we identified USP5 as an essential deubiquitinase that binds to Snail protein phosphorylated by MSK1. Based on the experimental data, in CRC, MSK1-Snail-USP5 axis can promote EMT and metastasis of CRC. Together, our findings provide potential biomarkers and novel therapeutic targets for further research in CRC.
© 2025. The Author(s).

Fibrillin-1 G234D mutation in the hybrid1 domain causes tight skin associated with dysregulated elastogenesis and increased collagen cross-linking in mice.

In Matrix Biology : Journal of the International Society for Matrix Biology on 1 February 2025 by Hossain, A. S., Clarin, M. T. R. D. C., et al.

Fibrillin-1, an extracellular matrix (ECM) protein encoded by the FBN1 gene, serves as a microfibril scaffold crucial for elastic fiber formation and homeostasis in pliable tissue such as the skin. Aside from causing Marfan syndrome, some mutations in FBN1 result in scleroderma, marked by hardened and thicker skin which limits joint mobility. Here, we describe a tight skin phenotype in the Fbn1G234D/G234D mice carrying a corresponding variant of FBN1 in the hybrid1 domain that was identified in a patient with familial aortic dissection. Unlike scleroderma, skin thickness and collagen fiber abundance do not change in the Fbn1G234D/G234D mutant skin. Instead, increased collagen cross-links were observed. In addition, short elastic fibers were sparsely located underneath the panniculus muscle layer, and an abundance of thin, aberrant elastic fibers was increased within the subcutaneous fascia, which may have tightened skin attachment to the underlying skeletal muscle. Structurally, Fbn1G234D/G234D microfibrils have a disrupted shoulder region that shares similarities with hybrid1 deletion mutant microfibrils. We then demonstrate the consequence of fibrillin-1 G234D mutation on dermal fibroblast functions. Mutant primary fibroblasts produce fewer elastic fibers, exhibit slower migration and increased cell stiffness. Moreover, secretome from mutant fibroblasts are marked by enhanced secretion of ECM, ECM-modifying enzymes, proteoglycans and cytokines, which are pro-tissue repair/fibrogenic. The transcriptome of mutant fibroblasts displays an increased expression of myogenic developmental and immune-related genes. Our study proposes that imbalanced ECM homeostasis due to a fibrillin-1 G234D mutation impacts fibroblast properties with potential ramifications on skin function.
Copyright © 2024. Published by Elsevier B.V.

Modulators of Alpha-2 Macroglobulin Upregulation by High Glucose in Glomerular Mesangial Cells.

In Biomolecules on 13 November 2024 by Trink, J., Li, R., et al.

Up to 40% of patients with diabetes mellitus will develop diabetic kidney disease (DKD), characterized pathologically by the accumulation of extracellular matrix proteins, which leads to the loss of kidney function over time. Our previous studies showed that the pan-protease inhibitor alpha 2-macroglobulin (A2M) is increased in DKD and is a critical regulator of the fibrotic response in glomerular mesangial cells (MC), an initial site of injury during DKD development. How A2M is regulated by high glucose (HG) has not yet been elucidated and is the focus of this investigation. Using serial deletions of the full A2M promoter, we identified the -405 bp region as HG-responsive in MC. Site-directed mutagenesis, siRNA, and ChIP studies showed that the transcription factor, nuclear factor of activated T cells 5 (NFAT5), regulated A2M promoter activity and protein expression in response to HG. Forkhead box P1 (FOXP1) served as a cooperative binding partner for NFAT5, required for A2M upregulation. Lastly, we showed that Smad3, known for its role in kidney fibrosis, regulated A2M promoter activity and protein production independently of HG. The importance of NFAT5, FOXP1, and Smad3 in A2M regulation was confirmed in ex vivo studies using isolated glomeruli. In conclusion, Smad3 is required for basal and HG-induced A2M expression, while NFAT5 and FOXP1 cooperatively regulate increased A2M transcription in response to HG. Inhibition of NFAT5/FOXP1 will be further evaluated as a potential therapeutic strategy to inhibit A2M production and attenuate profibrotic signaling in DKD.

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Chaperone-mediated autophagy modulates Snail protein stability: implications for breast cancer metastasis.

In Mol Cancer on 11 October 2024 by Ryu, K. J., Lee, K. W., et al.

Fig.4.E

Evasion of CMA-mediated Snail degradation induces EMT and increases metastatic abilities in luminal-type breast cancer cells. (A) Immunoblot analysis of NL-treated WT-Snail- or 58AAAA-Snail-expressing MCF-7 cells (upper). Relative Snail levels were quantified using ImageJ (lower). *, P < 0.05: **, P < 0.01: ***, P < 0.001 as determined by t-test. ns, not significant. (B) Immunoblot analysis in LAMP2A-depleted WT-Snail- or 58AAAA-Snail-expressing MCF-7 cells (left). Relative Snail levels were quantified using ImageJ (right). **, P < 0.01 as determined by t-test. ns, not significant. (C) WT-Snail or 58AAAA-Snail-expressing MCF-7 cells were treated with CHX (100 µg/ml) for the indicated times before harvest. Cell lysates were immunoblotted with the indicated antibodies (left), and data quantified using ImageJ (right). Normalized to α-tubulin. **, P < 0.01: ***, P < 0.001: ****, P < 0.0001 as determined by t-test. ns, not significant. (D) Morphological changes of WT-Snail or 58AAAA-Snail-expressing MCF-7 cells. WT-Snail-, 58AAAA-Snail-expressing MCF-7 and control cells were stained with TRITC-conjugated phalloidin and visualized by confocal microscopy. Scale bars, 40 μm–20 μm. (E) Expression levels of EMT marker proteins in WT-Snail-, 58AAAA-Snail-expressing MCF-7 and control cells were analyzed by immunoblotting. (F) WT-Snail, 58AAAA-Snail-expressing MCF-7 and control cells were analyzed in wound-healing assays by visualizing wound closure via phase-contrast microcopy (left). Wound areas were measured using WimScratch software (Wimasis). The data shown represent the percentage of the wound area and are expressed as the means ± SD of three individual experiments (right). **, P < 0.01: ****, P < 0.0001 as determined by t-test. (G) WT-Snail-, 58AAAA-Snail-expressing MCF-7 and control cells were seeded onto Matrigel matrix-coated top chambers, and the fold-changes of invading cells were measured after 30 h. The data shown are expressed as the means ± SD of three individual experiments, each performed in triplicate. **, P < 0.01: ****, P < 0.0001 as determined by t-test. (H) The indicated cells were seeded in a 6-well plate at a concentration of 1 × 105 cells per well. After incubation for 1 to 4 days, the viable cells were counted with a hemocytometer after trypan blue staining. (I) About 2 × 106 of the MCF-7 cells stably expressing WT-Snail, 58AAAA-Snail, or control cells were injected into the nude mice by tail-vein injection. Representative pictures of HE staining of lung sections (left). Scale bars, 200 μm. The number of metastatic lung nodules in individual mice was quantified at 6 weeks after tail-vein injection (right). The data are shown as the means ± SD of 7 mice/group. **, P < 0.01: ***, P < 0.001 as determined by t-test less

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NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension.

In JCI Insight on 23 April 2024 by Papaioannou, I., Dritsoula, A., et al.

Fig.2.A

NKX2-5 expression is associated with the synthetic phenotype in HPASMCs.HPASMCs were cultured in vitro under conditions favoring the contractile or synthetic phenotype over 7 days. (A) Protein expression levels of NKX2-5, synthetic cell markers COL1, and FN1, and contractile cell markers ACTA2, CNN1, and TAGLN were determined by Western blotting. A representative blot from 3 independent experiments is shown. Statistical significance was examined by 2-way ANOVA (Supplemental Figure 8). The effect of FBS and the effect of time were both significant at P < 0.05 for all genes. All genes come from the same samples run on different, but concurrent, blots, except for COL1, which was run on a separate occasion. (B) Nuclear extracts were used to quantify NKX2-5 expression via Western blotting with the E1Y8H antibody. A representative blot from 2 independent experiments is shown. Substantial expression of NKX2-5 was only observed after prolonged culture in serum (P < 0.05 via 2-tailed Student’s t test). NKX2-5 and TBP were run on different, but concurrent, blots. (C) Immunofluorescence was carried out on contractile (7 days in 1% FBS) or synthetic (7 days in 10% FBS) HPASMCs using specific antibodies for NKX2-5 (Alexa Fluor 488, green), intracellular pro-collagen type I (Procol1, Alexa Fluor 488, green), FN1 (Alexa Fluor 594, red), ACTA2 (Cy3, orange), and DAPI (blue). Nuclear expression of NKX2-5 was concomitant with high COL1 and FN1 expression in synthetic, but not contractile, HPASMCs. High levels of organized ACTA2 expression were only visible in contractile cells. Scale bars: 50 μm. All protein molecular weights are given as kDa in parentheses. less

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Reversion-inducing cysteine-rich protein with Kazal motifs and MT1-MMP promote the formation of robust fibrillin fibers.

In J Cell Physiol on 1 March 2021 by Matsuzaki, T., Keene, D. R., et al.

Fig.2.A

FBN1 and FN fibers produced in vitro by MDFs derived from wild‐type (WT) and RECK‐Hypo mice. (a) Confluent cultures of MDFs prepared from WT (a1) or Reck‐Hypo (a2) mice at Postnatal Day 3 were subjected to double immunofluorescence staining with anti‐FBN1 (red) and anti‐FN (green) antibodies followed by nuclear counterstaining (blue). Images of the same field in each color and the merged image are shown in four‐parts panels. Images of background staining (i.e., no primary antibodies) are also shown (a3). Scale bar = 10 μm. As compared to the control, FBN1 fibers produced by Reck‐Hypo MDFs (a2) tend to form local aggregates in the area where FN fibers are few (yellow arrows). Substantial FN signals are found in spotty structures in RECK‐Hypo MDFs (green signals in a2). (b and c) Properties of FBN1 and FN fibers. (b) Intensity of FBN1 (b1) and FN (b2) signals at peaks (P) and valleys (V). (b1) Number of data: WT P, 1,134 and WT V, 1,123; Hypo P, 1,149 and Hypo V, 1,139. Student's t test (WT vs. Hypo): P, p = .0015; V, p = .0013. (b2) Number of data: WT P, 1,148; WT V, 1,143; Hypo P, 1,122; Hypo V, 1,110. Student's t test (WT vs. Hypo): P, p = .094; V, p = .041. (c) Width of FBN1 fibers (c1) and FN fibers (c2). Welch's t test (WT vs. Hypo): FBN1, p = .043; FN, p = .13. Similar results were obtained from three different mice of each group. FN, fibronectin; MDF, mouse dermal fibroblast less

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Reversion-inducing cysteine-rich protein with Kazal motifs and MT1-MMP promote the formation of robust fibrillin fibers.

In J Cell Physiol on 1 March 2021 by Matsuzaki, T., Keene, D. R., et al.

Fig.6.A

FBN1 fibers produced in vitro by MT1‐KO MDFs. (a) Confluent cultures of control MDFs transfected with empty vector (a1) and MT1‐KO MDFs transfected with either empty vector (a2), MT1‐MMP‐expression vector (a3), or RECK‐expression vector (a3) were subjected to double immunofluorescence staining with anti‐FBN1 (red) and anti‐FN (green) antibodies followed by nuclear counterstaining (blue). Scale bar = 10 μm. Note the poor formation of both FBN1 and FN fibers in MT1‐KO MDFs (a2), the ability of MT1‐MMP‐expression vector to at least partially restore the formation of FBN1 and FN fibers (a3), and the ability of RECK to restore the formation of FBN1 fibers to some extent but not the formation of FN fibers (a4). Similar results were obtained in three independent experiments. (b and c) Properties of FBN1 and FN fibers. (b) The intensity of FBN1 (b1) and FN (b2) signals at peaks (left panels) and valleys (right panels). The number of data are as follows. FBN1‐Peaks: Cont. + Vector, 1,375; MT1‐KO + Vector, 1,426; MT1‐KO + MT1, 1,444; MT1‐KO + RECK, 1,302. FBN1‐Valleys: Cont. + Vector, 1,367; MT1‐KO + Vector, 1,416; MT1‐KO + MT1, 1,435; MT1‐KO + RECK, 1,292. FN‐Peaks: Cont. + Vector, 1,406; MT1‐KO + Vector, 1,460; MT1‐KO + MT1, 1,295; MT1‐KO + RECK, 1,472; FN‐Valleys: Cont. + Vector, 1,395; MT1‐KO + Vector, 1,453; MT1‐KO + MT1, 1,282; MT1‐KO + RECK, 1,462. (c) Width of FBN1 fibers (c1) and FN fibers (c2). Welch's t test: *p < .05, **p < 5 × 10−3, ***p < 5 × 10−10. Note that the mean of widths (indicated by X in c) is largely consistent with the above observations of immunofluorescence images (a). Similar results were obtained in three independent experiments. Cont. control; FN, fibronectin; KO, knockout; M, MT1‐MMP‐expression vector; MDF, mouse dermal fibroblast; MT1‐MMP, membrane‐type 1‐ matrix metalloproteinase; R, RECK‐expression vector; V, empty vector less

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Transglutaminase-2 facilitates extracellular vesicle-mediated establishment of the metastatic niche.

In Oncogenesis on 13 February 2020 by Shinde, A., Paez, J. S., et al.

Fig.6.C

Tensin-1 is required for fibronectin fibrillogenesis on EVs.a Comparison of overall survival between patients bearing grade 3 tumors expressing levels of TNS1 above (high) or below (low) the mean level for the entire patient cohort. Survival curves were analyzed via a log-rank test resulting in the indicated p value. b Immunoblot analyses of EVs collected from control (GFP) and TG2-overexpressing HME2 (TGM2) as well as control (shMT) and TG2-depleted (shTGM2) HME2-BM cells. Lysates from these EVs were assessed for the levels of TG2 and TNS1, and the levels of CD63 served as a loading control. c Immunoblot analyses of EVs derived from control (shscram) and Tns1-depleted (shTns1) 4T1 cells. Lysates from these EVs were assessed for the levels of TG2 and Tns1, and the levels of CD63 served as a loading control. d The fibrillar FN-specific (FN3) antibody was used in conjunction with immuno-electron microscopy to analyze nonpermeabilized EVs derived from control (shscram) and Tns1-depleted (shTns1) 4T1 cells. e Control (shscram) and Tns1-depleted (shTns1) 4T1 cells were grown under single-cell 3D culture conditions. Cellular outgrowth was quantified by bioluminescence at Day 8. Data are normalized to the plated values and are the mean ± SD of three independent analyses resulting in the indicated p value. f Control (shscram) and Tns1-depleted (shTns1) 4T1 cells were engrafted onto the mammary fat pad, and the resultant primary tumors were removed and weighed upon necropsy. g Quantification of bioluminescent radiance from the thoracic regions of interest (ROIs) at the indicated time points. h Upon necropsy, the numbers of pulmonary metastatic nodules were quantified. (Inset) Corresponding gross anatomical views of lungs from control (shscram) and Tns1-depleted (shTns1) 4T1 tumor-bearing mice. Data in f–h are mean ± SE values from five mice per group resulting in no significance (n.s.) or the indicated p values. less

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CD109 acts as a gatekeeper of the epithelial trait by suppressing epithelial to mesenchymal transition in squamous cell carcinoma cells in vitro.

In Sci Rep on 6 November 2019 by Zhou, S., da Silva, S. D., et al.

Fig.6.B

CD109 loss promotes EMT process, motility and invasion of SCCs. (A) Expression analysis by qPCR of fibronectin (FN), vimentin (VIM), a-SMA, Collagen 1A1 (Col1A1) and Collagen 3A1 (Col3A1). Results were first normalized with the housekeeping gene GAPDH, and the expressions in parental A431 cells were set at 1. Results are presented as mean ± SD (n = 3 independent experiments repeated in triplicates). (B) Representative Western blot image and (C) quantification of EMT markers. CD109 loss significantly enhanced the EMT marker expressions. (D) Representative images of Western blot and (E) Immunofluorescence microscopy and (F) quantification for P-Smad2 expression in A431 and CD109 KO A431 cells, showing an enhanced p-Smad2 activity in the CD109-KO cells. (G) Representative images and (H) quantification of wound-healing assays on parental and CD109 KO A431 cells, indicating CD109 loss enhanced the migration of SCC cells. Cell migration is expressed as a percentage of the scratch area filled by migrating cells at 24 h post scratch: migration rate = (T0 hr scratch width − T24 hr scratch width)/T0 hr scratch width) × 100%. (I) Representative images and (J) quantification of the invasive assay, suggesting CD109 loss significantly promoted cancer cell invasion in matrigel invasion assay. 50,000 cells of parental or CD109 KOA431 cells were seeded on a BioCoat™ Matrigel® Invasion Chamber for 24 hours. Cells that invaded through the matrigel-coated membrane were stained with 1% crystal violet, photographed, and counted. All the results are expressed as the mean ± S.D. of three independent experiments. Significance is calculated using a One-Way ANOVA *P < 0.05. **P < 0.01 and ***P < 0.001. Scale bars: 10 μM, 100 μM and 300 μM, as indicated. less

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