CiteAb

Fig.3.B showing Western Blotting from the publication: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring.

Fig.2.E showing Western Blotting from the publication: Mitochondrial uncoupler DNP induces coexistence of dual-state hyper-energy metabolism leading to tumor growth advantage in human glioma xenografts.

Fig.5.D showing Western Blotting from the publication: Mitochondrial uncoupler DNP induces coexistence of dual-state hyper-energy metabolism leading to tumor growth advantage in human glioma xenografts.

Fig.5.B showing Western Blotting from the publication: Mitochondrial uncoupler DNP induces coexistence of dual-state hyper-energy metabolism leading to tumor growth advantage in human glioma xenografts.

Fig.1.E showing Western Blotting from the publication: Mitochondrial uncoupler DNP induces coexistence of dual-state hyper-energy metabolism leading to tumor growth advantage in human glioma xenografts.

Fig.4.A showing Western Blotting from the publication: mTOR Contributes to the Proteome Diversity through Transcriptome-Wide Alternative Splicing.

Fig.3.B showing Western Blotting from the publication: Sweet taste receptor agonists attenuate macrophage IL-1β expression and eosinophilic inflammation linked to autophagy deficiency in myeloid cells.

Fig.6.B showing Western Blotting from the publication: Sweet taste receptor agonists attenuate macrophage IL-1β expression and eosinophilic inflammation linked to autophagy deficiency in myeloid cells.

Fig.5.E showing Western Blotting from the publication: p63, a key regulator of Ago2, links to the microRNA-144 cluster.

Fig.5.D showing Western Blotting from the publication: p63, a key regulator of Ago2, links to the microRNA-144 cluster.

Fig.4.D showing Western Blotting from the publication: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

Fig.5.D showing Western Blotting from the publication: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

Fig.5.A showing Western Blotting from the publication: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

Fig.2.C showing Western Blotting from the publication: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

Fig.2.D showing Western Blotting from the publication: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

Fig.7.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: In vivo monitoring of the recruitment and activation of AP-1 by Arf1.

Fig.9.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: In vivo monitoring of the recruitment and activation of AP-1 by Arf1.

Fig.1.B showing Western Blotting from the publication: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells.

Fig.2.E showing Western Blotting from the publication: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells.

Fig.4.A showing Western Blotting from the publication: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells.

Fig.6.C showing Western Blotting from the publication: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells.

Fig.7.A showing Western Blotting from the publication: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells.

Fig.7.E showing Western Blotting from the publication: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells.

Fig.8.E showing Western Blotting from the publication: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells.

Fig.8.B showing Western Blotting from the publication: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells.

38 images found

Disuse plasticity limits spinal cord injury recovery.

In IScience on 18 April 2025 by Morioka, K., Tazoe, T., et al.

Use-dependent plasticity after spinal cord injury (SCI) enhances neuromotor function, however, the optimal timing to initiate rehabilitation remains controversial. To test impacts of early disuse, we established a rodent model of transient hindlimb suspension in acute phase SCI. Early disuse in the first 2-week after SCI undermined recovery on open-field locomotion, kinematics, and swim tests even after 6-week of normal gravity reloading. Early disuse produced chronic spinal circuit hyper-excitability in H-reflex and interlimb reflex tests. Quantitative synaptoneurosome analysis of lumboventral spinal cords revealed shifts in AMPA receptor (AMPAR) subunit GluA1 localization and serine 881 phosphorylation, reflecting enduring synaptic memories of early disuse stored in the spinal cord. Automated confocal analysis of motoneurons revealed persistent shifts toward GluA2-lacking, calcium-permeable AMPARs in disuse subjects. Unsupervised machine learning associated multidimensional synaptic changes with persistent recovery deficits in SCI. The results argue for early aggressive rehabilitation to prevent disuse plasticity that limits SCI recovery.
© 2025 The Authors.

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PANoptosis, a combination of inflammatory cell death mechanisms, induced by Ophiobolin A in breast cancer cell lines

Preprint on BioRxiv : the Preprint Server for Biology on 14 April 2025 by Ranganathan, S., Ojo, T., et al.

An unmet challenge in managing breast cancer is treatment failure due to resistance to apoptosis-inducing chemotherapies. Thus, it is important to identify novel non-apoptotic therapeutic agents. Several non-apoptotic programmed cell death pathways utilize specific cellular signaling events to trigger lytic and pro-inflammatory cell death. PANoptosis, which encompasses pyroptosis, apoptosis and necroptosis, is of paramount importance in the regulation of cell death and immune responses. Our study illustrates that ophiobolin A (OpA) is an anti-cancer agent that triggers lytic cell death in breast cancer cells, including triple-negative breast cancer (TNBC), via a mechanism dependent on RIPK1. This study reveals that OpA induces typical pyroptosis-like characteristics, including cellular swelling, plasma membrane rupture, GSDMD cleavage and release of cytokines in breast cancer cells. The involvement of caspase 3, RIPK1, and GSDMD suggests that PANoptosis is activated upon OpA treatment in breast cancer. The induction of pro-inflammatory cell death suggests potential applications for OpA in cancer treatment.

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Mutant huntingtin induces neuronal apoptosis via derepressing the non-canonical poly(A) polymerase PAPD5.

In Nature Communications on 9 April 2025 by Chen, Z. S., Peng, S. I., et al.

MicroRNAs (miRNAs) are small non-coding RNAs that play crucial roles in post-transcriptional gene regulation. Poly(A) RNA polymerase D5 (PAPD5) catalyzes the addition of adenosine to the 3' end of miRNAs. In this study, we demonstrate that the Yin Yang 1 protein, a transcriptional repressor of PAPD5, is recruited to both RNA foci and protein aggregates, resulting in an upregulation of PAPD5 expression in Huntington's disease (HD). Additionally, we identify a subset of PAPD5-regulated miRNAs with increased adenylation and reduced expression in our disease model. We focus on miR-7-5p and find that its reduction causes the activation of the TAB2-mediated TAK1-MKK4-JNK pro-apoptotic pathway. This pathway is also activated in induced pluripotent stem cell-derived striatal neurons and post-mortem striatal tissues isolated from HD patients. In addition, we discover that a small molecule PAPD5 inhibitor, BCH001, can mitigate cell death and neurodegeneration in our disease models. This study highlights the importance of PAPD5-mediated miRNA dysfunction in HD pathogenesis and suggests a potential therapeutic direction for the disease.
© 2025. The Author(s).

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Host WD repeat-containing protein 5 inhibits protein kinase R-mediated integrated stress response during measles virus infection.

In Journal of Virology on 17 September 2024 by BenDavid, E., Yang, C., et al.

Some negative-sense RNA viruses, including measles virus (MeV), share the characteristic that during their infection cycle, cytoplasmic inclusion bodies (IBs) are formed where components of the viral replication machinery are concentrated. As a foci of viral replication, how IBs act to enhance the efficiency of infection by affecting virus-host interactions remains an important topic of investigation. We previously established that upon MeV infection, the epigenetic host protein, WD repeat-containing protein 5 (WDR5), translocates to cytoplasmic viral IBs and facilitates MeV replication. We now show that WDR5 is recruited to IBs by forming a complex with IB-associated MeV phosphoprotein via a conserved binding motif located on the surface of WDR5. Furthermore, we provide evidence that WDR5 promotes viral replication by suppressing a major innate immune response pathway, the double-stranded RNA-mediated activation of protein kinase R and integrated stress response.
MeV is a pathogen that remains a global concern, with an estimated 9 million measles cases and 128,000 measles deaths in 2022 according to the World Health Organization. A large population of the world still has inadequate access to the effective vaccine against the exceptionally transmissible MeV. Measles disease is characterized by a high morbidity in children and in immunocompromised individuals. An important area of research for negative-sense RNA viruses, including MeV, is the characterization of the complex interactome between virus and host occurring at cytoplasmic IBs where viral replication occurs. Despite the progress made in understanding IB structures, little is known regarding the virus-host interactions within IBs and the role of these interactions in promoting viral replication and antagonizing host innate immunity. Herein we provide evidence suggesting a model by which MeV IBs utilize the host protein WDR5 to suppress the protein kinase R-integrated stress response pathway.

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Oligodendrocyte-derived laminin-γ1 regulates the blood-brain barrier and CNS myelination in mice.

In Cell Reports on 28 May 2024 by Kang, M. & Yao, Y.

Although oligodendrocytes (OLs) synthesize laminin-γ1, the most widely used γ subunit, its functional significance in the CNS remains unknown. To answer this important question, we generated a conditional knockout mouse line with laminin-γ1 deficiency in OL lineage cells (γ1-OKO). γ1-OKO mice exhibit weakness/paralysis and die by post-natal day 33. Additionally, they develop blood-brain barrier (BBB) disruption in the cortex and striatum. Subsequent studies reveal decreased major facilitator superfamily domain containing 2a expression and increased endothelial caveolae vesicles, but unaltered tight junction protein expression and tight junction ultrastructure, indicating a transcellular, rather than a paracellular, mechanism of BBB breakdown. Furthermore, significantly reduced OL lineage cells, OL precursor cells (OPCs), proliferating OPCs, and mature OLs are observed in γ1-OKO brains in a region-specific manner. Consistent with this finding, various defects in myelination are detected in γ1-OKO brains at biochemical and ultrastructural levels. Overall, these results highlight important roles of OL-derived laminin-γ1 in BBB maintenance and OL biology (proliferation, differentiation, and myelination).
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

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Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring.

In Cells on 24 January 2024 by Chatterjee, B., Fatima, F., et al.

Fig.3.B

Fig.3.B showing Western Blotting from the publication: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring.

Generation of in vivo knockdown models of CBS and CGL, regulators of transsulfuration pathway involved in Hcy catabolism. (A) Simplified diagram showing the enzymes and metabolites of transsulfuration pathway (B) Representative western blot of 2 dpf embryos lysates showing that compared with SC M... more

Generation of in vivo knockdown models of CBS and CGL, regulators of transsulfuration pathway involved in Hcy catabolism. (A) Simplified diagram showing the enzymes and metabolites of transsulfuration pathway (B) Representative western blot of 2 dpf embryos lysates showing that compared with SC MO injected embryos, CBS protein level is downregulated in CBS MO injected embryos. Expression of CGL, the other enzyme of the same pathway, remained unaltered. As a loading control β-actin was used. Corresponding bar plot showing fold change in protein expression (normalized to β-actin) as determined by densitometric analysis of the protein band. (C) Bar plot revealing no statistically significant difference in the viability of scrambled control and Hyperhomocysteinemic CBS MO embryos. (D) Bright field images exhibiting no apparent gross morphological defect in CBS morphants compared with scrambled Mo injected embryos of 2 dpf. Scale bar, 0.25 mm. (E) Representative western blot of embryo lysates at 2 dpf showing reduced protein level of CGL in the embryos injected with CGL MO. Expression of CBS, the upstream protein of the same pathway, remained unaltered. β-actin was used as a loading control. Corresponding bar diagram showing densitometric analysis of the fold change in protein expression (normalized to β-actin). (F) In comparison to SC MO injected embryos, viability of CGL MO injected embryos were not altered significantly. (G) Representative bright field images showing absence of any gross morphological defect in CGL MO injected embryos compared with SC MO injected ones at 2 dpf. Scale bar, 0.25 mm. Data are shown as Mean ± SEM with n ≥ 3. ** p ≤ 0.01, **** p ≤ 0.0001 and ns is non-significant (p > 0.05). less

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Mitochondrial uncoupler DNP induces coexistence of dual-state hyper-energy metabolism leading to tumor growth advantage in human glioma xenografts.

In Front Oncol on 3 January 2023 by Rai, Y., Singh, S., et al.

Fig.2.E

Fig.2.E showing Western Blotting from the publication: Mitochondrial uncoupler DNP induces coexistence of dual-state hyper-energy metabolism leading to tumor growth advantage in human glioma xenografts.

DNP modulates mitochondrial respiration. (A) OCR was quantified as the value of MitoXpressXtra fluorescence lifetime signal (μs·/min./μg protein) with or without FCCP (3 μM) in BMG-1 and OPM-BMG cells (n=3). (B) The overall change in the OCR showed by OPM-BMG cells is presented as % a relative ch... more

DNP modulates mitochondrial respiration. (A) OCR was quantified as the value of MitoXpressXtra fluorescence lifetime signal (μs·/min./μg protein) with or without FCCP (3 μM) in BMG-1 and OPM-BMG cells (n=3). (B) The overall change in the OCR showed by OPM-BMG cells is presented as % a relative change with respect to BMG-1 (derived information from graph A). (C) ATP content of both BMG-1 and OPM-BMG cells were determined at 24 h post-seeding by ATP bioluminescence assay method, and obtained values were compared among the groups and presented as pmol/cell (n=3). (D) Mitochondrial ROS and mitochondrial mass (Mito-mass) were determined using MitoSox Red and MitoTracker Green, respectively. Column graph showing an increase in mean fluorescence intensity (MFI) as a relative change for both fluorescent probes in OPM-BMG cells with respect to BMG-1 cells. (E) Immunoblotting of indicated mitochondrial proteins depicting the difference in expression level and densitometric evaluation plotted as relative change among both the cell lines. (F) Metabolic viability was evaluated as a mitochondrial function of cell lines by MTT assay (n=4) and presented as a change in MTT value (ΔOD, y-axis) with respect to time (x-axis). Data are presented as the mean ± SD; P values (BMG-1 versus OPM-BMG cells) are mentioned in the graphs. less

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mTOR Contributes to the Proteome Diversity through Transcriptome-Wide Alternative Splicing.

In Int J Mol Sci on 17 October 2022 by Cheng, S., Fahmi, N. A., et al.

Fig.4.A

Fig.4.A showing Western Blotting from the publication: mTOR Contributes to the Proteome Diversity through Transcriptome-Wide Alternative Splicing.

mTOR-driven exon skipping in CDS confers multifarious features in the proteome. (A) Analysis of protein isoform stability by Western blotting. Flag-tagged SIRT2 protein isoforms were transiently expressed in HEK293 and the difference in their stabilities was monitored in the presence of cyclohexi... more

mTOR-driven exon skipping in CDS confers multifarious features in the proteome. (A) Analysis of protein isoform stability by Western blotting. Flag-tagged SIRT2 protein isoforms were transiently expressed in HEK293 and the difference in their stabilities was monitored in the presence of cycloheximide (30 ug/mL) for the indicated time points. Actin was used as a loading control. The protein level was quantified using densitometry in ImageStudioLite software and normalized to the actin level. Means (SD) from two technical repeats were subjected to two-tailed Student’s t-test for statistical analysis. p < 0.05 as significant (*). n.s. denotes no significance. (B) Proportion of mTOR-regulated AS from the coding region showed a Pfam domain change. (C) Examples of affected Pfam domains and their associated GO terms by mTOR-driven AS events. Bioinformatics analysis of the functional Pfam domains disrupted or gained by AS events in the CDS of mRNA was performed. Pink: Pfam domains gained in hyper-activated mTOR. Light purple: Pfam domains lost in hyper-activated mTOR. Green: Pfam domains affected in both low and high mTOR environments. Rectangle: molecular functions; diamond: biological processes; triangle: cellular pathways. (D) Various post-translational modification (PTM) sites found on protein isoforms generated by mTOR-regulated AS. Protein isoforms produced by four types of AS are presented from top to bottom. Four types of PTM (phosphorylation, acetylation, succinylation, and ubiquitination) were interrogated using published mass spectrometry datasets. The number of unique PTM sites found in either exon-included or exon-skipped protein isoforms is summed. The number of common PTM sites located in both protein isoforms is also reported. (E) Two examples of published unique functional phosphorylation events found on mTOR-regulated AS protein isoforms of SIRT2 and ARF2. less

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Sweet taste receptor agonists attenuate macrophage IL-1β expression and eosinophilic inflammation linked to autophagy deficiency in myeloid cells.

In Clin Transl Med on 1 August 2022 by Lee, J., Kim, S. J., et al.

Fig.6.B

Fig.6.B showing Western Blotting from the publication: Sweet taste receptor agonists attenuate macrophage IL-1β expression and eosinophilic inflammation linked to autophagy deficiency in myeloid cells.

Human T1R3 in association with T1R2 contribute to the suppression of IL‐1β production. (A) Relative mRNA levels of T1R3 in THP‐1 cells or PBMC from two different donors were determined by using PCR (left) or qRT‐PCR (right) with normalisation to β‐actin mRNA. (B) Relative protein levels of T1R3 i... more

Human T1R3 in association with T1R2 contribute to the suppression of IL‐1β production. (A) Relative mRNA levels of T1R3 in THP‐1 cells or PBMC from two different donors were determined by using PCR (left) or qRT‐PCR (right) with normalisation to β‐actin mRNA. (B) Relative protein levels of T1R3 in THP‐1 cells or PBMC from healthy donor were determined by immunoblotting with β‐actin control. (C) Effect of trehalose (left) or saccharin (right) on the levels of IL‐1β in the supernatants of activated PBMC from healthy donors, as determined by ELISA. (D) Effect of T1R3 overexpression in the absence or the presence of T1R2 on trehalose (left)‐ or saccharin (right)‐mediated inhibition of IL‐1β levels in activated THP‐1 cells, as determined by ELISA. (E) Effect of U73122, U73433, and SQ22536 on trehalose‐ or saccharin‐mediated suppression of IL‐1β levels in activated THP‐1 cells, as determined by ELISA. (F) Effect of trehalose (left) or saccharin (right) on the levels of IL‐1β in the supernatants of activated PBMC from patient with chronic rhinosinusitis (CRS), as determined by ELISA. Data are expressed as means ± SDs. *P < .05, **P < .01, and ***P < .005, Student's t‐test. Data are representative of at least three independent experiments performed in duplicate or triplicate less

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Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

In Nat Commun on 25 March 2021 by Alabi, A., Xia, X. D., et al.

Fig.4.D

Fig.4.D showing Western Blotting from the publication: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

Metabolic effects of MT1LKO mice.a MT1-MMP deletion. Whole-cell lysate of primary mouse hepatocytes isolated from MT1Flox and MT1LKO mice was subjected to immunoblotting. Similar results were obtained from three independent experiments. b, c Relative expression of target genes. qRT-PCR measuremen... more

Metabolic effects of MT1LKO mice.a MT1-MMP deletion. Whole-cell lysate of primary mouse hepatocytes isolated from MT1Flox and MT1LKO mice was subjected to immunoblotting. Similar results were obtained from three independent experiments. b, c Relative expression of target genes. qRT-PCR measurement of mRNA levels of MT1-MMP in different tissues (b) (four or five mice per group) or MT1-MMP, MT2-MMP, and Adam17 in the liver (five or six mice per group) (c). The relative mRNA levels were the ratio of the mRNA levels of the target genes from different tissues to that of Gapdh in the same tissue. WAT, white adipose tissue. d Liver LDLR levels (6 mice per group). Liver homogenate was subjected to immunoblotting. Relative densitometry was the ratio of the densitometry of LDLR of different mice to that of actin of the same mouse. Representative images were shown. Similar results were obtained from the other three mice. e Plasma sLDLR determined by ELISA (6 mice per group). f Relative mRNA levels (4 mice per group). g Plasma levels of total cholesterol (6 mice per group). h Lipid profile. Same amount of plasma from each mouse in the same group was pooled and applied to FPLC analysis of plasma cholesterol (6 mice per group). i–k Expression of human MT1-MMP. MT1Flox and MT1LKO mice were injected with AAVs encoding GFP or human MT1-MMP, respectively. Liver homogenate was applied to immunoblotting (6 mice per group). Representative images were shown. Similar results were obtained from the other three mice (i). Plasma samples were used to measure sLDLR levels (6 mice in the control group and 5 mice in MT1-MMP overexpression group) (j), and total cholesterol levels (6 mice per group) (k). Student’s t test (two-sided) was carried out to determine the significant differences between groups (b–g, j and k). The significance was defined as p < 0.05. Values of all data were mean ± SD. Source data are provided as a Source Data file. less

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Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

In Nat Commun on 25 March 2021 by Alabi, A., Xia, X. D., et al.

Fig.5.D

Fig.5.D showing Western Blotting from the publication: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

The impact of MT1-MMP.a Overexpression of human MT1-MMP. Male C57BL/6J mice were injected with empty or the wild-type MT1-MMP adenoviruses (5 mice per group). Liver homogenate was subject to immunoblotting, followed by quantification of LDLR levels relative to actin in the same mouse. Representat... more

The impact of MT1-MMP.a Overexpression of human MT1-MMP. Male C57BL/6J mice were injected with empty or the wild-type MT1-MMP adenoviruses (5 mice per group). Liver homogenate was subject to immunoblotting, followed by quantification of LDLR levels relative to actin in the same mouse. Representative images were shown. Similar results were obtained from other mice. Plasma levels of HDL (b) and non-HDL cholesterol (c) (4 mice per group). d Effect of the Western-type diet (6 mice per group). Mice were fed the Western-type diet for 8 weeks. Liver homogenate was subjected to immunoblotting. Relative densitometry of LDLR was determined as described. Actin was used as a loading control. Representative images were shown. Similar results were obtained from the other mice. Liver section staining. Representative figures and quantification (ImageJ 1.52S) of Masson’s Trichrome (e) and oil Red-O staining (f) in cross-sections of the liver (6 mice per group). Similar results were obtained from the other mice (magnification: 400×). Plasma levels of cholesterol content in HDL (g) and non-HDL (h) (4 mice per group). i Lipid profile. Same amount of plasma from each mouse in the same group (2 female and 2 male mice per group) was pooled and applied to FPLC analysis of plasma cholesterol. Student’s t test (two-sided) was carried out to determine the significant differences between groups (a–h). The significance was defined as p < 0.05. Values of all data were mean ± SD. Source data are provided as a Source Data file. less

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Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

In Nat Commun on 25 March 2021 by Alabi, A., Xia, X. D., et al.

Fig.5.A

Fig.5.A showing Western Blotting from the publication: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

The impact of MT1-MMP.a Overexpression of human MT1-MMP. Male C57BL/6J mice were injected with empty or the wild-type MT1-MMP adenoviruses (5 mice per group). Liver homogenate was subject to immunoblotting, followed by quantification of LDLR levels relative to actin in the same mouse. Representat... more

The impact of MT1-MMP.a Overexpression of human MT1-MMP. Male C57BL/6J mice were injected with empty or the wild-type MT1-MMP adenoviruses (5 mice per group). Liver homogenate was subject to immunoblotting, followed by quantification of LDLR levels relative to actin in the same mouse. Representative images were shown. Similar results were obtained from other mice. Plasma levels of HDL (b) and non-HDL cholesterol (c) (4 mice per group). d Effect of the Western-type diet (6 mice per group). Mice were fed the Western-type diet for 8 weeks. Liver homogenate was subjected to immunoblotting. Relative densitometry of LDLR was determined as described. Actin was used as a loading control. Representative images were shown. Similar results were obtained from the other mice. Liver section staining. Representative figures and quantification (ImageJ 1.52S) of Masson’s Trichrome (e) and oil Red-O staining (f) in cross-sections of the liver (6 mice per group). Similar results were obtained from the other mice (magnification: 400×). Plasma levels of cholesterol content in HDL (g) and non-HDL (h) (4 mice per group). i Lipid profile. Same amount of plasma from each mouse in the same group (2 female and 2 male mice per group) was pooled and applied to FPLC analysis of plasma cholesterol. Student’s t test (two-sided) was carried out to determine the significant differences between groups (a–h). The significance was defined as p < 0.05. Values of all data were mean ± SD. Source data are provided as a Source Data file. less

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Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

In Nat Commun on 25 March 2021 by Alabi, A., Xia, X. D., et al.

Fig.2.C

Fig.2.C showing Western Blotting from the publication: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

MT1-MMP-mediated LDLR cleavage.a Inhibitors treatment. Huh7.5 cells transfected with empty pCDNA3.1 or HA-tagged MT1-MMP-pCDNA3.1were incubated with MG132 (MG) or chloroquine (Chloro). Whole-cell lysate was subjected to immunoblotting. HA-tagged MT1-MMP was detected by an anti-HA antibody. b MT1-... more

MT1-MMP-mediated LDLR cleavage.a Inhibitors treatment. Huh7.5 cells transfected with empty pCDNA3.1 or HA-tagged MT1-MMP-pCDNA3.1were incubated with MG132 (MG) or chloroquine (Chloro). Whole-cell lysate was subjected to immunoblotting. HA-tagged MT1-MMP was detected by an anti-HA antibody. b MT1-MMP knockdown. qRT-PCR of Huh7.5 cells transfected with scrambled or MT1-MMP siRNAs. The relative mRNA levels were the ratio of the mRNA levels of the target genes to that of GAPDH at the same condition. The fold-change of the relative mRNA levels of target gene expression in MT1-MMP siRNA treated groups was determined in comparison with that in the control group that was defined as 1. c MT1-MMP overexpression. Cells as indicated were infected with either empty (Emp), the wild-type (WT), or mutant E240A (E240A) MT1-MMP adenoviruses. Whole-cell lysate was subjected to immunoblotting. d LDLR cleavage (n = 3 independent experiments). Huh7.5 cells transfected with scrambled (Scram) or MT1-MMP siRNA (MT1-1) were cultured in DMEM only medium for 16 h. Whole-cell lysate (bottom) and concentrated media (top) were subjected to immunoblotting. MT1: MT1-MMP; Alb, albumin; TFR, transferrin receptor. e Ectodomain cleavage (n = 3 independent experiments). HEK293 cells were transfected with plasmid indicated. After 36 h, culture medium was changed to DMEM containing 0.5% FBS for overnight. Whole-cell lysate (bottom) and culture medium (top) were subjected to immunoblotting. The bottom figures in panels d and e showed representative protein levels. The relative densitometry was the ratio of the densitometry of sLDLR to that of albumin in culture medium in the same sample. Soluble LDLR. Culture medium was collected from Huh7.5 cells (f) (n = 4 independent experiments) or HEK293 cells (g) (n = 3 independent experiments) and subjected to measurement of sLDLR using ELISA. Similar results were obtained from at least three independent experiments. Student’s t test (two-sided) was carried out to determine the significant differences between groups (d–g). The significance was defined as p < 0.05. Values of all data were mean ± SD. Source data are provided as a Source Data file. less

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Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

In Nat Commun on 25 March 2021 by Alabi, A., Xia, X. D., et al.

Fig.2.D

Fig.2.D showing Western Blotting from the publication: Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis.

MT1-MMP-mediated LDLR cleavage.a Inhibitors treatment. Huh7.5 cells transfected with empty pCDNA3.1 or HA-tagged MT1-MMP-pCDNA3.1were incubated with MG132 (MG) or chloroquine (Chloro). Whole-cell lysate was subjected to immunoblotting. HA-tagged MT1-MMP was detected by an anti-HA antibody. b MT1-... more

MT1-MMP-mediated LDLR cleavage.a Inhibitors treatment. Huh7.5 cells transfected with empty pCDNA3.1 or HA-tagged MT1-MMP-pCDNA3.1were incubated with MG132 (MG) or chloroquine (Chloro). Whole-cell lysate was subjected to immunoblotting. HA-tagged MT1-MMP was detected by an anti-HA antibody. b MT1-MMP knockdown. qRT-PCR of Huh7.5 cells transfected with scrambled or MT1-MMP siRNAs. The relative mRNA levels were the ratio of the mRNA levels of the target genes to that of GAPDH at the same condition. The fold-change of the relative mRNA levels of target gene expression in MT1-MMP siRNA treated groups was determined in comparison with that in the control group that was defined as 1. c MT1-MMP overexpression. Cells as indicated were infected with either empty (Emp), the wild-type (WT), or mutant E240A (E240A) MT1-MMP adenoviruses. Whole-cell lysate was subjected to immunoblotting. d LDLR cleavage (n = 3 independent experiments). Huh7.5 cells transfected with scrambled (Scram) or MT1-MMP siRNA (MT1-1) were cultured in DMEM only medium for 16 h. Whole-cell lysate (bottom) and concentrated media (top) were subjected to immunoblotting. MT1: MT1-MMP; Alb, albumin; TFR, transferrin receptor. e Ectodomain cleavage (n = 3 independent experiments). HEK293 cells were transfected with plasmid indicated. After 36 h, culture medium was changed to DMEM containing 0.5% FBS for overnight. Whole-cell lysate (bottom) and culture medium (top) were subjected to immunoblotting. The bottom figures in panels d and e showed representative protein levels. The relative densitometry was the ratio of the densitometry of sLDLR to that of albumin in culture medium in the same sample. Soluble LDLR. Culture medium was collected from Huh7.5 cells (f) (n = 4 independent experiments) or HEK293 cells (g) (n = 3 independent experiments) and subjected to measurement of sLDLR using ELISA. Similar results were obtained from at least three independent experiments. Student’s t test (two-sided) was carried out to determine the significant differences between groups (d–g). The significance was defined as p < 0.05. Values of all data were mean ± SD. Source data are provided as a Source Data file. less

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