Journal: American Journal of Translational Research

Article Title: CLOCK disruption aggravates carotid artery stenosis through endoplasmic reticulum stress-induced endothelial-mesenchymal transition

doi:

Figure Lengend Snippet: The ClockΔ19/Δ19 mutation exacerbated endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in vivo. A. p-PERK, p-eIF2α, p-IRE1α, XBP-1, and ATF6 protein levels in partially ligated CAs assayed by western blotting (n = 6). B. Immunofluorescence staining for CD31 (green) and XBP-1 (red). Nuclei were stained with DAPI (blue). Scale bar: 100 μm. C. Immunocytochemical analysis of the percentage of XBP-1+ ECs in the intima of partially ligated CAs (n = 6). D. Immunofluorescence intensity of XBP-1 in the intima of partially ligated CAs (n = 6). *P < 0.05, **P < 0.01.

Article Snippet: Anti-ATF6 (ab37149) and anti-GAPDH (ab8245) antibodies were obtained from Abcam (Cambridge, MA, USA), anti-Xbp-1 (24385) was from Signalway Antibody (Baltimore, USA), anti-p-IRE1α (Ser724; {"type":"entrez-protein","attrs":{"text":"ARG40603","term_id":"1175807486","term_text":"ARG40603"}} ARG40603 ) and anti-p-eIF2α (Ser51; {"type":"entrez-protein","attrs":{"text":"ARG57722","term_id":"1176876255","term_text":"ARG57722"}} ARG57722 ) were from arigo Biolaboratories Corp (Taiwan), and anti-p-PERK (Thr980; 3179s) was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Mutagenesis, In Vivo, Western Blot, Immunofluorescence, Staining

Journal: American Journal of Translational Research

Article Title: CLOCK disruption aggravates carotid artery stenosis through endoplasmic reticulum stress-induced endothelial-mesenchymal transition

doi:

Figure Lengend Snippet: ClockΔ19/Δ19 aggravated EndMT and endoplasmic reticulum (ER) stress induced by disturbed flow (DF) in vitro. A. Relative mRNA levels of Cdh5, S100a4, Acta2, Vimentin, Twist1, Snai1, Mmp2, and Mmp9 in pMAECs exposed to disturbed flow (DF) or undisturbed flow (UF) (n = 3). B, C. p-PERK, p-eIF2α, p-IRE1α, XBP-1, and ATF6 protein levels of pMAECs exposed to disturbed flow (DF) or undisturbed flow (UF) assayed by western blotting (n = 3). *P < 0.05 vs. WT+DF, **P < 0.01 vs. WT+DF, #P < 0.05 vs. WT+UF, ##P < 0.01 vs. WT+UF.

Article Snippet: Anti-ATF6 (ab37149) and anti-GAPDH (ab8245) antibodies were obtained from Abcam (Cambridge, MA, USA), anti-Xbp-1 (24385) was from Signalway Antibody (Baltimore, USA), anti-p-IRE1α (Ser724; {"type":"entrez-protein","attrs":{"text":"ARG40603","term_id":"1175807486","term_text":"ARG40603"}} ARG40603 ) and anti-p-eIF2α (Ser51; {"type":"entrez-protein","attrs":{"text":"ARG57722","term_id":"1176876255","term_text":"ARG57722"}} ARG57722 ) were from arigo Biolaboratories Corp (Taiwan), and anti-p-PERK (Thr980; 3179s) was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vitro, Western Blot

Journal: American Journal of Translational Research

Article Title: CLOCK disruption aggravates carotid artery stenosis through endoplasmic reticulum stress-induced endothelial-mesenchymal transition

doi:

Figure Lengend Snippet: ClockΔ19/Δ19 (Clk) mutation aggravated disturbed flow (DF)-induced EndMT by activating the IRE1α-XBP1 axis. Wild-type (WT) or ClockΔ19/Δ19 pMAECs were exposed to disturbed flow (DF) and DMSO (WT or Clk), 30 µΜ STF-083010 (Clk+S), 15 µΜ STF-083010 (Clk+S’), 0.03 μM GSK2606414 (Clk+G), or 0.015 μM GSK2606414 (Clk+G’) for 72 h. A-D. Cdh5, S100a4, Vimentin, and Snai1 mRNA levels were assayed by quantitative reverse transcription PCR (n = 3). *P < 0.05 vs. WT, **P < 0.01 vs. WT, # P < 0.05 vs. Clk, ## P < 0.01 vs. Clk.

Article Snippet: Anti-ATF6 (ab37149) and anti-GAPDH (ab8245) antibodies were obtained from Abcam (Cambridge, MA, USA), anti-Xbp-1 (24385) was from Signalway Antibody (Baltimore, USA), anti-p-IRE1α (Ser724; {"type":"entrez-protein","attrs":{"text":"ARG40603","term_id":"1175807486","term_text":"ARG40603"}} ARG40603 ) and anti-p-eIF2α (Ser51; {"type":"entrez-protein","attrs":{"text":"ARG57722","term_id":"1176876255","term_text":"ARG57722"}} ARG57722 ) were from arigo Biolaboratories Corp (Taiwan), and anti-p-PERK (Thr980; 3179s) was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Mutagenesis

Journal: American Journal of Translational Research

Article Title: CLOCK disruption aggravates carotid artery stenosis through endoplasmic reticulum stress-induced endothelial-mesenchymal transition

doi:

Figure Lengend Snippet: The mechanism for the inhibitory effect of CLOCK as a suppressor of carotid plaque progression. Under disturbed flow, the disturbance of endothelial CLOCK expression activates the IRE1α-XBP1 axis and then leads to endothelial-to-mesenchymal transition; this results in increased inflammation. The results of our research showed that CLOCK attenuated carotid plaque stenosis and vulnerable plaque progression through the signaling pathways above.

Article Snippet: Anti-ATF6 (ab37149) and anti-GAPDH (ab8245) antibodies were obtained from Abcam (Cambridge, MA, USA), anti-Xbp-1 (24385) was from Signalway Antibody (Baltimore, USA), anti-p-IRE1α (Ser724; {"type":"entrez-protein","attrs":{"text":"ARG40603","term_id":"1175807486","term_text":"ARG40603"}} ARG40603 ) and anti-p-eIF2α (Ser51; {"type":"entrez-protein","attrs":{"text":"ARG57722","term_id":"1176876255","term_text":"ARG57722"}} ARG57722 ) were from arigo Biolaboratories Corp (Taiwan), and anti-p-PERK (Thr980; 3179s) was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing

Journal: Experimental & Molecular Medicine

Article Title: The natural product salicin alleviates osteoarthritis progression by binding to IRE1α and inhibiting endoplasmic reticulum stress through the IRE1α-IκBα-p65 signaling pathway

doi: 10.1038/s12276-022-00879-w

Figure Lengend Snippet: a Heatmap for global gene expression with group clusters ( n = 3). b Volcano map of DEGs in the SA group vs. the control group (upregulation: 75 genes and downregulation: 32 genes). DEGs with FC (fold change) ≥ 2 were accepted as positive DEGs. c Pathway enrichment bubble map based on the KEGG enrichment analysis. A rich factor indicates a higher degree of enrichment, a larger p value (−log10) indicates a higher statistical significance, and a larger bubble indicates a higher degree of enrichment. d GO enrichment of those DEGs. A rich factor indicates a higher degree of enrichment, a larger p value (−log10) indicates a higher statistical significance, and a larger bubble indicates a higher degree of enrichment, BP: biological process, CC: cellular component. e Molecular docking for estimating the binding site of IRE1α and SA. The three-dimensional (3D) structures of IRE1α and SA were subjected to molecular docking, and the binding site was obtained according to the software scoring system. Upper left panel: SA was located in the cavity formed by the IRE1α 3D structure, upper right panel: magnification of the cavity; lower left panel: binding site of SA and IRE1α, lower left panel: magnification showing the hydrogen bonds (yellow dot line) formed between SA and IRE1α. f DARTS assay for confirmation of IRE1α and SA binding. Protease was used to digest IRE1α protein. With the addition of SA, the digestion of IRE1α was significantly blocked at each concentration of protease compared with the DMSO group.

Article Snippet: Then, the sections were incubated overnight at 4 °C with primary phospho-NF-κB p65 (CST), Ki-67 (D3B5) (CST) and p-IRE1α (Zen Bio) antibodies.

Techniques: Expressing, Binding Assay, Software, Concentration Assay

Journal: Experimental & Molecular Medicine

Article Title: The natural product salicin alleviates osteoarthritis progression by binding to IRE1α and inhibiting endoplasmic reticulum stress through the IRE1α-IκBα-p65 signaling pathway

doi: 10.1038/s12276-022-00879-w

Figure Lengend Snippet: a WB analysis for detecting IRE1α and downstream gene expression at the protein level. The ER stress-associated proteins GRP78, pIRE1α, IRE1α, p-IκBα, t-IκBα, p-p65 and p65 were detected in each group (i). Quantitative analysis of GRP78 (ii), the ratio of p-IRE1α/t-IRE1α (iii), the ratio of p-IκBα/t-IκBα (iv), and the ratio of p-p65/t-p-p65 (v) at the protein level. GAPDH was used as a reference protein ( n = 3, one-way ANOVA). b P65 nuclear translocation in each treatment group. IF was used to detect p-65 in the nucleus, and DAPI was used to stain the cell nucleus; scale bar: 50 μm. c The IRE1α inhibitor APY29 blocked TNF-α-mediated ER stress. Chondrocytes were stimulated with TNF-α and then treated with 10 μM SA or 10 μM APY29 for 48 h. XPB1 u and XPB1 s were detected by RT-QPCR (i), and XPB1s, MMP13, GRP78, IRE1α, p-IRE1α, p-p65 and p65 were detected by WB analysis (ii). Quantitative analysis of XPB1s (iii), MMP13 (iv), GRP78 (v), the ratio of p-IRE1α/t-IRE1α (vi), and the ratio of p-p65/t-p65 (vii) at the protein level. GAPDH was used as a reference protein ( n = 3, one-way ANOVA). The data are expressed as the mean ± SD, * p < 0.05, ** p < 0.01, * ** p < 0.001, and ns, not significant.

Article Snippet: Then, the sections were incubated overnight at 4 °C with primary phospho-NF-κB p65 (CST), Ki-67 (D3B5) (CST) and p-IRE1α (Zen Bio) antibodies.

Techniques: Expressing, Translocation Assay, Staining, Quantitative RT-PCR

Journal: Experimental & Molecular Medicine

Article Title: The natural product salicin alleviates osteoarthritis progression by binding to IRE1α and inhibiting endoplasmic reticulum stress through the IRE1α-IκBα-p65 signaling pathway

doi: 10.1038/s12276-022-00879-w

Figure Lengend Snippet: a P-IRE1α in chondrocytes in each treatment group. IF was used to detect p-IRE1α in each treatment group. p-IRE1α was highly expressed in the cytoplasm of chondrocytes in the ACLT and vehicle groups, and SA dramatically decreased the expression of p-IRE1α in the cytoplasm of chondrocytes (i). Quantitative analysis showed that ACLT-induced high expression of p-IRE1α was significantly reversed by SA treatment (ii) ( n = 5, one-way ANOVA). b P-p65 nuclear translocation in each treatment group. ACLT-induced p-p65 nuclear translocation was ameliorated by SA treatment (i). Quantitative analysis of each treatment group showed the same trend (ii). Scale bar, 50 μm; the ACLT group indicates the group injected with PBS; the vehicle group indicates the group injected with only PLGA vehicle; the SA group indicates the group injected with SA-loaded PLGA. Dashed lines indicate the cartilage surface. The data are expressed as the mean ± SD, * p < 0.05, ** p < 0.01, * ** p < 0.001, and ns, not significant. c A proposed model of action. SA binding to IRE1α blocked IRE1α phosphorylation and inhibited IRE1α-mediated ER stress via IRE1α-IκBα-p65 signaling.

Article Snippet: Then, the sections were incubated overnight at 4 °C with primary phospho-NF-κB p65 (CST), Ki-67 (D3B5) (CST) and p-IRE1α (Zen Bio) antibodies.

Techniques: Expressing, Translocation Assay, Injection, Binding Assay