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

Journal: Scientific Reports

Article Title: SHP2 improves ovarian morphology and steroidogenic function in a rat PCOS model by modulating IRE1α/XBP1/NLRP3-mediated granulosa cell pyroptosis

doi: 10.1038/s41598-026-43536-2

Figure Lengend Snippet: A . Boxplot for data standardization evaluation. B . Principal component analysis scatter plot. C . Volcano plot of differentially expressed genes. D . Heatmap of differentially expressed genes. Ea. GO cellular component (CC) enrichment bubble plot. Eb. GO molecular function (MF) enrichment bubble plot. Ec. GO biological process (BP) enrichment bubble plot. Fa. KEGG pathway enrichment bubble plot. Fb. KEGG pathway enrichment lollipop plot. G . Correlation pie chart of key regulatory factors (IRE1α, XBP-1, p38, SP1, ZEB1, PKP3, Rb, E2F1, Cyclin, SHP2, BRD4). H . Scatter plot of E2F1-CDK1 co-regulation. I. Scatter plot of NLRP3-GSDMD pyroptosis axis.

Article Snippet: Antibodies against p-SHP2, p-IRE1α, XBP-1s, p-SP1, ZEB1, PKP3, nuclear E2F1, CyclinB1, NLRP3, GSDMD, and GAPDH were purchased from Wuhan Sanying Biotechnology Co., Ltd. IgG secondary antibodies and ECL hypersensitive luminescent solution were purchased from Beijing Biosynthesis Biotechnology Co., Ltd.

Techniques:

Journal: Scientific Reports

Article Title: SHP2 improves ovarian morphology and steroidogenic function in a rat PCOS model by modulating IRE1α/XBP1/NLRP3-mediated granulosa cell pyroptosis

doi: 10.1038/s41598-026-43536-2

Figure Lengend Snippet: SHP2 Alleviates PCOS Phenotypes via IRE1α/XBP1/NLRP3 and ZEB1/PKP3-Mediated Regulation. (A) Western blot detection of SHP2-regulated IRE1α/XBP1/ZEB1/PKP3 protein pathways. (B) Quantitative analysis of protein expression levels.* p < 0.05, ** p < 0.01, ns indicates P > 0.05; data are represented as the mean ± SD.

Article Snippet: Antibodies against p-SHP2, p-IRE1α, XBP-1s, p-SP1, ZEB1, PKP3, nuclear E2F1, CyclinB1, NLRP3, GSDMD, and GAPDH were purchased from Wuhan Sanying Biotechnology Co., Ltd. IgG secondary antibodies and ECL hypersensitive luminescent solution were purchased from Beijing Biosynthesis Biotechnology Co., Ltd.

Techniques: Western Blot, Expressing

Journal: Scientific Reports

Article Title: SHP2 improves ovarian morphology and steroidogenic function in a rat PCOS model by modulating IRE1α/XBP1/NLRP3-mediated granulosa cell pyroptosis

doi: 10.1038/s41598-026-43536-2

Figure Lengend Snippet: SHP2 improves ovarian morphology and steroidogenic function in a rat PCOS model by regulating the IRE1α/XBP1/NLRP3 and ZEB1/PKP3 signaling pathways, thereby influencing granulosa cell pyroptosis and proliferation.

Article Snippet: Antibodies against p-SHP2, p-IRE1α, XBP-1s, p-SP1, ZEB1, PKP3, nuclear E2F1, CyclinB1, NLRP3, GSDMD, and GAPDH were purchased from Wuhan Sanying Biotechnology Co., Ltd. IgG secondary antibodies and ECL hypersensitive luminescent solution were purchased from Beijing Biosynthesis Biotechnology Co., Ltd.

Techniques: Protein-Protein interactions