Journal: bioRxiv

Article Title: Salicin modifies osteoarthritis progression by binding on IRE1α and inhibiting IRE1α mediated endoplasmic reticulum stress

doi: 10.1101/2022.04.06.487311

Figure Lengend Snippet: (A) Molecular docking for estimating combination site of IRE1α and SA. Three-dimensional (3D) structure of IRE1α and SA were subjected for molecular docking, combination site was obtained according to the software scoring system. Upper left panel: SA was located in the cavity formed by IRE1α 3D structure, upper right panel: magnification of the cavity; lower left panel: combination site of SA and IRE1α, lower left panel: magnification shown the hydrogen bonds (yellow dot line) formed between SA and IRE1α. (B) DARTS assay for confirmation of IRE1α and SA combination, 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 DMSO group.(C) Illustrative live-cell fluorescence microscopy images of ER damge labeled with ER-Tracker Red, chondrocytes exposed to TNF-α were treated with or without SA, ER damage was stained with red (upper panel), cell nucleus wree stained with DAPI (middle panel) and merged images were shown (lower panel).(scale bar: 50 μm).

Article Snippet: After blocking with 5% skimmed milk, membranes were probed with primary antibodies against Col2α1 (Abcam, ab34712), Aggrecan (Abcam, ab3773), MMP13 (Zen Bio, 820098), BCL2 (Zen Bio, R22494), CDK1 (Abcam, ab134175), total-ERK1/2 (CST, #4695), phospho-ERK1/2 (CST, #4370), total-IκBα (CST, #4812), phospho-IκBα (CST, #2859), phospho-IRE1α (Zen Bio, R26310), IRE1α (bs-16696R), GRP78 (Zen bio, R24509), NF-κB p65 antibody (CST,#8242), Phospho-NF-κB p65 antibody (CST, #3033), GAPDH (Zen Bio, 380626) overnight at 4 °C.

Techniques: Software, Concentration Assay, Fluorescence, Microscopy, Labeling, Staining

Journal: bioRxiv

Article Title: Salicin modifies osteoarthritis progression by binding on IRE1α and inhibiting IRE1α mediated endoplasmic reticulum stress

doi: 10.1101/2022.04.06.487311

Figure Lengend Snippet: SA regulates IRE1α mediated ER stress by IRE1α-IκBα-p65 signaling. (A) WB analysis for detecting IRE1α and downstream genes expression at protein level. ER stress associated protein GRP78, pIRE1α, IRE1α, p-IκBα, t-IκBα, p-p65 and p65 were detected in each group (a). Quantitative analysis of GRP78 (b), ratio of p-IRE1α/t-IRE1α (c), ratio of p-IκBα/t-IκBα (d) and ratio of p-p65/t-p-p65 (e) at protein level, GAPDH was used as reference protein (n=3, one-way ANOVA). (B) P65 nucleus translocation in each treatment group. IF was used to detect the p-65 in nucleus, DAPI was used to staining cell nucleus, scale bar: 50 μm. (C) ER stress inhibitor 4-PBA blocked TNF-α mediated ER stress. Chondrocytes were stimulated with TNF-α and then treated with 10 μM SA or 15 μM 4-PBA for 48 h, MMP13, GRP78, IRE1α, p-IRE1α, p-p65 and p65 were detected by WB analysis (a). Quantitative analysis of MMP13 (b), GRP78 (c), ratio of p-IRE1α/t-IRE1α (d), and ratio of p-p65/t-p65 (e) at protein level, GAPDH was used as reference protein (n=3, one-way ANOVA). The data are expressed as mean ± SD, *p<0.05, **p<0.01, ***p <0.001, and ns, not significant.

Article Snippet: After blocking with 5% skimmed milk, membranes were probed with primary antibodies against Col2α1 (Abcam, ab34712), Aggrecan (Abcam, ab3773), MMP13 (Zen Bio, 820098), BCL2 (Zen Bio, R22494), CDK1 (Abcam, ab134175), total-ERK1/2 (CST, #4695), phospho-ERK1/2 (CST, #4370), total-IκBα (CST, #4812), phospho-IκBα (CST, #2859), phospho-IRE1α (Zen Bio, R26310), IRE1α (bs-16696R), GRP78 (Zen bio, R24509), NF-κB p65 antibody (CST,#8242), Phospho-NF-κB p65 antibody (CST, #3033), GAPDH (Zen Bio, 380626) overnight at 4 °C.

Techniques: Expressing, Translocation Assay, Staining

Journal: bioRxiv

Article Title: Salicin modifies osteoarthritis progression by binding on IRE1α and inhibiting IRE1α mediated endoplasmic reticulum stress

doi: 10.1101/2022.04.06.487311

Figure Lengend Snippet: SA intra-articular injection ameliorates ACLT induced OA progression by inhibiting IRE1α mediated ER stress. (A) Diagram summarized the animal experiments. ACLT was used for the construction of knee OA model. Four weeks later, intra-articular injection of SA-loaded PLGA was done, and intra-articular injection of PLGA only was used as control. Four weeks after treatment, rats in each group were sacrificed and knee joints were subjected to histological analysis respectively. (B) H&E and Safranin-O/green staining for each treatment group. H&E (upper panel) and Safranin-O/green staining (lower panel) showed that ACLT resulted in the thicker of cartilage and degeneration of cartilage matrix compared with sham group, and intra-articular injection of SA-loaded PLGA ameliorated this progression compared with vehicle (PLGA only) group or sham group. OARSI scoring (b) and cartilage matrix area quantitative analysis (c) showed that, although OARSI score in ACLT, vehicle and SA groups were statistically higher than sham group, OARSI score in SA group significantly lower than ACLT and vehicle groups (n=5, one-way ANOVA). (C) IHC for detecting cartilage matrix degeneration MMP13, ER stress marker GRP78 and p-IRE1α, NC, negative control, scale bar 50 μm. (D) P-p65 nucleus translocation in each treatment group, scale bar 50 μm. (E) Quantitative analysis showed that the expression of MM13 (a), GRP78 (b) and p-IRE1α (c) in ACLT group or vehicle group were significantly more than that in sham group, and the expression of MMP13, GRP78 and p-IRE1α in SA group were significantly less than those in ACLT group and vehicle group (n=5, one-way ANOVA). Quantitative analysis showed that P-p65 nucleus translocation in ACLT group or vehicle group was significantly more than that in sham group, and p-p65 nucleus translocation in SA group was obviously less than that in ACLT group and vehicle group (d) (n=5, random fields, one-way ANOVA). ACLT group indicates the group with PBS injection; Vehicle group indicates only inject PLGA vehicle; SA group indicates injection of SA loaded PLGA. Dash lines indicate cartilage surface. The data are expressed as mean ±SD, *p<0.05, **p<0.01, ***p<0.001, and ns, not significant.

Article Snippet: After blocking with 5% skimmed milk, membranes were probed with primary antibodies against Col2α1 (Abcam, ab34712), Aggrecan (Abcam, ab3773), MMP13 (Zen Bio, 820098), BCL2 (Zen Bio, R22494), CDK1 (Abcam, ab134175), total-ERK1/2 (CST, #4695), phospho-ERK1/2 (CST, #4370), total-IκBα (CST, #4812), phospho-IκBα (CST, #2859), phospho-IRE1α (Zen Bio, R26310), IRE1α (bs-16696R), GRP78 (Zen bio, R24509), NF-κB p65 antibody (CST,#8242), Phospho-NF-κB p65 antibody (CST, #3033), GAPDH (Zen Bio, 380626) overnight at 4 °C.

Techniques: Injection, Staining, Marker, Negative Control, Translocation Assay, Expressing

Journal: bioRxiv

Article Title: Salicin modifies osteoarthritis progression by binding on IRE1α and inhibiting IRE1α mediated endoplasmic reticulum stress

doi: 10.1101/2022.04.06.487311

Figure Lengend Snippet: A proposed model of action. SA bound on IRE1α and blocked IRE1α phosphorylation, then inhibits IRE1α mediated ER stress by IRE1α-IκBα-p65 signaling.

Article Snippet: After blocking with 5% skimmed milk, membranes were probed with primary antibodies against Col2α1 (Abcam, ab34712), Aggrecan (Abcam, ab3773), MMP13 (Zen Bio, 820098), BCL2 (Zen Bio, R22494), CDK1 (Abcam, ab134175), total-ERK1/2 (CST, #4695), phospho-ERK1/2 (CST, #4370), total-IκBα (CST, #4812), phospho-IκBα (CST, #2859), phospho-IRE1α (Zen Bio, R26310), IRE1α (bs-16696R), GRP78 (Zen bio, R24509), NF-κB p65 antibody (CST,#8242), Phospho-NF-κB p65 antibody (CST, #3033), GAPDH (Zen Bio, 380626) overnight at 4 °C.

Techniques:

Journal: eLife

Article Title: Caspase-mediated cleavage of IRE1 controls apoptotic cell commitment during endoplasmic reticulum stress

doi: 10.7554/eLife.47084

Figure Lengend Snippet:

Article Snippet: Antibody , pIRE1α (rabbit monoclonal) , Genentech ( ) , , (1:500).

Techniques: Knock-Out, Isolation, Clone Assay, CRISPR, Transfection, Construct, FLAG-tag, shRNA, Recombinant, Caspase-Glo Assay, Cell Viability Assay, Flow Cytometry, Fractionation, Cell Culture, Purification, Software

Journal: Nature Communications

Article Title: Fortilin binds IRE1α and prevents ER stress from signaling apoptotic cell death

doi: 10.1038/s41467-017-00029-1

Figure Lengend Snippet: Fortilin selectively inhibits IRE1α signaling to protect against ER stress-induced apoptosis. a EGF-SubA dose-dependently cleaves GRP78 in PC3 cells. Wild-type PC3 cells were treated with the indicated concentrations of EGF-SubA, and their lysates were subjected to IB. b EGF-SubA treatment does not change the expression levels of fortilin in PC3 cells. PC3 sh-Empty and PC3 sh-Fortilin cells were treated with the indicated concentrations of EGF-SubA for 24 h and subjected to IB. c Three branches of ER stress signaling pathways. IRE1α, PERK, and ATF6 are the ER resident transmembrane proteins that signal ER stress when activated. IRE1α and PERK, but not ATF6, are known to induce apoptosis. d Fortilin does not regulate the PERK and ATF6 signaling pathways. PC3 sh-Empty and PC3 sh-Fortilin cells were treated with the indicated concentrations of EGF-SubA, and their lysates were subjected to quantitative IB for PERK, its downstream molecule eIF2α, and fragmented and activated ATF6 (ATF6f). Data were expressed as means ± s.d. ( n = 3) and analyzed by two-tailed unpaired t -test. * P < 0.05; ** P < 0.01; *** P < 0.005. e Fortilin inhibits the IRE1α ER stress signaling pathway. PC3 sh-Empty and PC3 sh-Fortilin cells were treated with the indicated concentrations of EGF-SubA and subjected to IB for IRE1α and its downstream molecules JNK and spliced XBP1 (XBP1s). P-IRE1α is the activated form of IRE1α, and P-JNK is the activated form of JNK. Data were expressed as means ± s.d. ( n = 3, except for P-IRE1α and XBP1s where n = 2) and analyzed by two-tailed unpaired t -test. * P < 0.05; ** P < 0.01; *** P < 0.005. f Fortilin inhibits the processing by IRE1α of XBP1 mRNA. PC3 sh-Empty and PC3 sh-Fortilin cells were treated with the indicated concentrations of EGF-SubA. The cDNA from total RNA from these cells was subjected to PCR and Pst1 restriction digestion (CTGCA|G) to semi-quantitatively evaluate the levels of XBP1s . Data were expressed as means ± s.d. ( n = 2) and analyzed by two-tailed unpaired t -test. * P < 0.05. XBP1u unspliced XBP1

Article Snippet: The system was tested using anti-fortilin antibody (positive control, Abnova) and bovine serum albumin (negative control, Sigma-Aldrich) as depicted in Supplementary Fig. . Next, various concentrations of recombinant human phopho-IRE1α-Myc-DDK (aa 1–977, Origene), de-phosphorylated IRE1α-Myc-DDK (aa 1–977, Origene, see above for the dephosphorylation procedure), GST-IRE1α-L (aa 1–70, Abnova), GST-IRE1α-TM (aa 401–500, Abnova), and GST-IRE1α-C (aa 468–977, SignalChem, Richmond, British Columbia, Canada) were added for 180 s to evaluate the association between the two molecules.

Techniques: Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Fortilin binds IRE1α and prevents ER stress from signaling apoptotic cell death

doi: 10.1038/s41467-017-00029-1

Figure Lengend Snippet: Fortilin interacts with the cytosolic domain of IRE1α and inhibits its protein kinase and RNase activities. a Proximity ligation assay (PLA) shows a specific interaction between fortilin and P-IRE1α in EGF-SubA-treated PC3 cells. The cells were treated with 2 nM EGF-SubA for 24 h and subjected to PLA, using anti-IREα and anti-P-IRE1α antibodies to evaluate fortilin-IRE1α and fortilin-P-IRE1α interaction, respectively. PLA interaction indices were calculated by dividing the number of red dots by the number of nuclei, expressed as means ± s.d. ( n = 3), and analyzed by two-tailed unpaired t -test. NS not statistically significant; *** P < 0.005. Scale bar = 10 µm. b Fortilin co-immunoprecipitates P-IRE1α. PC3 cells were treated with 2 nM EGF-SubA for 24 h, lysed and subjected to immunoprecipitation (IP). c Domain structure of human IRE1α. Human IRE1α consists of the ER luminal domain (aa 1–443), transmembrane domain (aa 444–464), linker region (aa 465–567), kinase domain (aa 568–833), and endoribonuclease (RNase) domain (aa 836–997). The following recombinant proteins were used for biolayer interferometry: full-length IRE1α (aa 1-977), IRE1α-Myc-DDK (aa 1–977); IRE1α-L, GST-IRE1α (aa 1–70); IRE1α-TM, GST-IRE1α (aa 401–500); and IRE1α-C, GST-IRE1α (aa 468–977). d – h Fortilin binds to P-IRE1α through its cytosolic domain. Biotinylated fortilin was immobilized to the streptavidin biosensor. Recombinant IRE1α, either full-length or fragment, was applied to the biosensor at various concentrations, and dissociation constants (Kds, expressed as mean ± s.d., n = 3) were derived. i Lowest energy binding pose of fortilin ( blue ) with cytosolic domain of IRE1α ( green ) (the right panel ) presented with that of a fortilin-fortilin dimer (the left panel ). j Intermolecular interactions between phosphorylated serine724 (pS 724 ) and serine726 (pS 726 ) of the cytosolic domain of IRE1α with lysine residues (K 19 and K 34 ) of fortilin. k Fortilin inhibits the RNase activity of IRE1α. An in vitro IRE1α RNase activity assay was performed by incubating IRE1α with human recombinant fortilin and the substrate fluorescently tagged XBP1 RNA stem loop, the cleavage of which would allow the fluorescein amidite (FAM) to fluoresce. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. *** P < 0.005. l Fortilin inhibits the kinase activity of IRE1α. An in vitro IRE1α kinase activity assay was performed by incubating IRE1α with [γ- 33 P]ATP, recombinant fortilin, and myelin basic protein (MBP) as a substrate of the kinase in the kinase reaction buffer. The phosphorylation index was calculated by dividing the radioactivity of MBP for a given fortilin concentration by that of the vehicle control and expressed as means ± s.d. ( n = 2) from which half maximal inhibitory concentration (IC 50 ) was calculated

Article Snippet: The system was tested using anti-fortilin antibody (positive control, Abnova) and bovine serum albumin (negative control, Sigma-Aldrich) as depicted in Supplementary Fig. . Next, various concentrations of recombinant human phopho-IRE1α-Myc-DDK (aa 1–977, Origene), de-phosphorylated IRE1α-Myc-DDK (aa 1–977, Origene, see above for the dephosphorylation procedure), GST-IRE1α-L (aa 1–70, Abnova), GST-IRE1α-TM (aa 401–500, Abnova), and GST-IRE1α-C (aa 468–977, SignalChem, Richmond, British Columbia, Canada) were added for 180 s to evaluate the association between the two molecules.

Techniques: Proximity Ligation Assay, Two Tailed Test, Immunoprecipitation, Recombinant, Derivative Assay, Binding Assay, Activity Assay, In Vitro, Kinase Assay, Radioactivity, Concentration Assay, Control

Journal: Nature Communications

Article Title: Fortilin binds IRE1α and prevents ER stress from signaling apoptotic cell death

doi: 10.1038/s41467-017-00029-1

Figure Lengend Snippet: Fortilin protects the whole animal against ER stress-induced liver failure and death by negatively regulating the IRE1α stress sensor pathway. a Liver-specific absence of fortilin in fortilin KO-liver mice. *, non-specific bands. b Experimental protocol. i.p. intraperitoneally, MOA mechanism of action, CMP complete metabolic panel, CBC complete blood count, IHC immunohistochemistry. c Higher survival rates of fortilin WT-liver than fortilin KO-liver mice after EGF-SubA challenge. A Kaplan–Meier survival curve and log-rank test showed the significantly better survival of fortilin WT-liver mice than that of fortilin KO-liver mice ( n = 10, P < 0.001). d Massive liver damage of EGF-SubA-treated fortilin KO-liver mice. The sera from fortilin WT-liver and fortilin KO-liver mice, treated with either PBS or EGF-SubA, were assayed for alanine aminotransferase (ALT; n = 5 and 10, for PBS and EGF-SubA, respectively), total bilirubin (TBIL; n = 3 and 6), and alkaline phosphatase (ALP; n = 3 and 6). Data were expressed as mean and analyzed by two-tailed unpaired t -test. NS not statistically significant; * P < 0.05; *** P < 0.005. e Drastic gross pathological change in the liver of EGF-SubA-treated fortilin KO-liver mice. Scale bar = 10 mm. f Fortilin fails to negatively regulate the PERK and ATF6 signaling pathways in the EGF-SubA-challenged liver. The total lysates from the livers of fortilin WT-liver and fortilin KO-liver mice, after the indicated treatments, were subjected to IB. Data were expressed as means ± s.d. ( n = 3) and analyzed by two-tailed unpaired t -test. NS not statistically significant. g Fortilin inhibits the IRE1α signaling pathway in the EGF-SubA-challenged liver. Data were expressed as means ± s.d. ( n = 3) and analyzed by two-tailed unpaired t -test. NS not statistically significant; ** P < 0.01. h Fortilin blocks IRE1α-mediated splicing of XBP1 mRNA in the liver of mice under ER stress. The amounts of XBP1s and XBP1u were quantified by quantitative densitometry. Data were expressed as means ± s.d. ( n = 5) and analyzed by two-tailed unpaired t -test. *** P < 0.005. i Fortilin inhibits the expression of XBP1s-inducible genes in the liver of mice under ER stress. Expression levels of ER-associated degradation (ERAD) genes— Edem1 and Herp1 —in the liver of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice were quantified by RT-qPCR with normalization against 18S rRNA. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. * P < 0.05, ** P < 0.01. j Fortilin blocks apoptosis and IRE1α pathway activation in EGF-SubA-challenged liver. Paraffin sections from the livers of EGF-SubA-treated fortilin WT-liver and fortilin KO-liver mice were subjected to TUNEL, P-IRE1α, and P-JNK staining using 3,3’-diaminobenzidine (DAB) as a chromogen. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. *** P < 0.005. Scale bar = 50 µm

Article Snippet: The system was tested using anti-fortilin antibody (positive control, Abnova) and bovine serum albumin (negative control, Sigma-Aldrich) as depicted in Supplementary Fig. . Next, various concentrations of recombinant human phopho-IRE1α-Myc-DDK (aa 1–977, Origene), de-phosphorylated IRE1α-Myc-DDK (aa 1–977, Origene, see above for the dephosphorylation procedure), GST-IRE1α-L (aa 1–70, Abnova), GST-IRE1α-TM (aa 401–500, Abnova), and GST-IRE1α-C (aa 468–977, SignalChem, Richmond, British Columbia, Canada) were added for 180 s to evaluate the association between the two molecules.

Techniques: Immunohistochemistry, Two Tailed Test, Expressing, Quantitative RT-PCR, Activation Assay, TUNEL Assay, Staining

Journal: Nature Communications

Article Title: Fortilin binds IRE1α and prevents ER stress from signaling apoptotic cell death

doi: 10.1038/s41467-017-00029-1

Figure Lengend Snippet: Protection by fortilin against EGF-SubA-induced liver damage is mediated by its ability to block the activation of the IRE1α-JNK apoptosis pathway. a Experimental protocol. Five-week-old male fortilin WT-liver or fortilin KO-liver mice ( n = 6) were pretreated by vehicle or the IRE1α kinase inhibiting RNAse attenuator-6 (KIRA6) for 3 days, challenged by EGF-SubA once on the third day, and treated by vehicle or KIRA6 for an additional 6 days. b KIRA6 protects both fortilin WT-liver and fortilin KO-liver mice against EGF-SubA-induced liver damage. The sera from EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice, treated with either vehicle or KIRA6, were assayed for ALT. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. NS not statistically significant; *** P < 0.005. c The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit similar gross appearance when treated with KIRA6. Scale bar = 10 mm. d Lack of change in expression and phosphorylation patterns of the PERK and ATF6 pathway proteins in the EGF-SubA-challenged livers, regardless of the status of fortilin or of KIRA6 treatment. The total lysates from the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice, treated with either KIRA6 or vehicle, were subjected to quantitative IB using the indicated antibodies. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. e The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit a similar degree of IRE1α and JNK phosphorylation when treated with KIRA6. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. * P < 0.05; *** P < 0.005. f The livers of EGF-SubA-challenged, KIRA6-treated, fortilin WT-liver and fortilin KO-liver mice exhibit a similar degree of XBP1 splicing. The total RNA from the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice were assayed for the amounts of XBP1s and XBP1u . Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. *** P < 0.005. g The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit equal amounts of apoptosis when treated with KIRA6. Paraffin sections from the livers of EGF-SubA-treated fortilin WT-liver and fortilin KO-liver mice ( n = 6 each) for cleaved lamin. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. ** P < 0.01; F.C . fold change. Scale bar = 50 µm. h , i The immunostaining of the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit the same degree of IRE1α pathway activation when treated with KIRA6. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. *** P < 0.005. Scale bar = 50 µm. j Proposed model of the role of fortilin in ER stress-induced apoptosis

Article Snippet: The system was tested using anti-fortilin antibody (positive control, Abnova) and bovine serum albumin (negative control, Sigma-Aldrich) as depicted in Supplementary Fig. . Next, various concentrations of recombinant human phopho-IRE1α-Myc-DDK (aa 1–977, Origene), de-phosphorylated IRE1α-Myc-DDK (aa 1–977, Origene, see above for the dephosphorylation procedure), GST-IRE1α-L (aa 1–70, Abnova), GST-IRE1α-TM (aa 401–500, Abnova), and GST-IRE1α-C (aa 468–977, SignalChem, Richmond, British Columbia, Canada) were added for 180 s to evaluate the association between the two molecules.

Techniques: Blocking Assay, Activation Assay, Two Tailed Test, Expressing, Immunostaining

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