anti ncor1 primary antibody  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) IHC-Alcian blue images showing NCoR1staining (brown colour) analysed in colon tissue biopsies sections of healthy individuals (n=5) and UC patients(n=5) (Scale bar= 100 µm). The inset shows zoomed-in areas of the image. Alcian blue (blue colour) represents colonic goblet cells and Nuclear fast red (red colour) represents nuclei. b) qRT-PCR analysis of relative fold expression of NCoR1 gene in human UC patient colonic biopsies (n=20) relative to average control value (n=24). 18s was used for normalisation. c) Representative immunoblot of NCoR1 protein in human ulcerative colitis (UC) (n=11) and control (n=9) biopsy samples. GAPDH was used as a loading control. Graph on the right showing densitometric analysis of NCoR1 expression normalised to control GAPDH. Each dot represents (B, C) one human. All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. See also Fig S1 and Table S1.

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) Schematic representation of the experimental plan for DSS dynamics in C57Bl/6 mice. Point of DSS administration indicated by red arrows (Created with BioRender.com ). b) Graph shows percent body weight change in Control, DSS3, DSS5 and DSS7mice groups. c) Gross morphology of colon and caeca of respective mice groups. The graph on the right shows colon length quantification. d) Immunoblot of NCoR1 protein in whole tissue lysate of DSS dynamics Control, DSS3, DSS5 and DSS7 mice. The graph on the right represents densitometric analysis showing fold intensity of NCoR1 expression calculated by normalising to loading control (GAPDH). e) Immunoblot of NCoR1 protein in crypt lysate Control, DSS1, DSS3 and DSS5. The graph on the right represents densitometric analysis showing fold intensity of NCoR1 expression calculated by normalising to loading control (Actin). f) Representative immunohistochemistry images of colon from control, DSS3,DSS5 and DSS7 mice stained for NCoR1 (n=3 per group) using anti-NCoR1 antibody. (scale bar=100um) .Zoom in images are shown in the inset. Each dot represents (c, d, e) one mouse. All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. See also Fig S2, Fig S3

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Control, Western Blot, Expressing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) Schematic representation of the experimental plan for chronic colitis in IL10 -/- mice using alternate DSS treatment (Point of administration indicated by red arrows) of 0.5% DSS in drinking water treatment for 12 weeks. (Created with BioRender.com ) b) Graph showing the difference in body weight of control and IL10 -/ mice. c) Gross morphology of colon and caeca of control and IL10 -/- mice. The graph on the right shows colon length quantification. d) Representative immunoblot of NCoR1 protein in whole tissue lysate of Control and IL10 -/- mice. Graph represents densitometric analysis showing the intensity of NCoR1 expression calculated by normalizing to loading control (GAPDH). Each dot represents (b, c and d) individual mice. All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. e) Haematoxylin Eosin staining in the distal colon tissue sections of control and IL10 -/- mice (n=3 per group) (scale bar= 50um).

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Control, Western Blot, Expressing, Staining

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) Schematic representation of the experimental plan for galactose-mediated HT29 pluripotent cells into goblet cells differentiation.(Created with BioRender.com ). b) Brightfield images showing goblet cell morphology in HT29 Undiff and HT29 Diff cells (Scale bar =50um). c) qRT -PCR analysis of relative fold expression of LGR5, MUC2 and MUC5AC genes in HT29 Diff cells with respect to HT29 undiff cells as control. HPRT was used for normalisation. Experiment performed in triplicate. d) Immunoblotting of NCoR1 and CLCA1 in HT29 Undiff and HT29 Diff cells. The graph on right represents densitometric analysis showing fold intensity of NCoR1 and CLCA1 expression calculated by normalising to loading control (GAPDH) e) Brightfield images showing goblet cell morphology in HT29 undiff-C , HT29 Undiff-kd , HT29 Diff-C and HT29 Diff-kd cells. Superscript C represents nontargeted knockdown control and superscript kd represents NCoR1 stable knockdown. Scale bar =50um. f) Representative Confocal images of NCoR1 (red) using anti-NCoR1 antibody and MUC2 (green) using anti-MUC2 antibody in HT29 undiff-C , HT29 Undiff-kd , HT29 Diff-C and HT29 Diff-kd cells (n=90) (scale bar=2 um). Graphs on the right shows NCoR1 and MUC2 fluorescence intensity measured. Each dot represents (d) individual experiment. All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. See also Fig S4

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot, Knockdown, Fluorescence

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) qRT-PCR analysis of relative fold expression of NCoR1 genes in HT29 Diff cells, with respect to control HT29 undiff cells. HPRT was used for normalization. b) Confocal imaging of MUC2 (green) stained using anti-MUC2 and NCoR1 (red) stained using anti-NCoR1 antibodies in HT29 MTX NCoR1kd andHT29 MTX scr . (scale bar=3um). Graphs on the right show MUC2 fluorescence intensity measured. c) Immunoblot of NCoR1expression in J774, HT29 Undiff ,HT29 Diff , HCT8 and HT29-MTX cell lines. Graph represents NCoR1 expression calculated by normalizing to loading control (Actin) in respective cell line. Each dot represents (a,b and c) individual experiment. All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Quantitative RT-PCR, Expressing, Control, Imaging, Staining, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) Schematic representation of the experimental plan for knockdown of NCoR1 in C57Bl/6 mice using vivo-morpholino w/c or w/o 2% DSS in drinking water. Point of administration of DSS (Red) control morpholino (blue) and NCoR1 Morpholino(green) is indicated by respective arrows. Morpholino(s) were administered via intraperitoneal injection on the mentioned point of administration (Created with BioRender.com ). b) Graph shows percentage body weight change in the mice groups Ctrl, DSS, Ctrl NCoR1kd and DSS NCoR1kd . c) Gross morphology of colon and caeca of indicated mice group. Graph on the right shows colon length quantification. d) Gross morphology of spleen of indicated mice group. Graph on the right shows spleen to body weight ratio. e) qRT-PCR analysis of relative fold expression of NCoR1 and MUC2 genes in DSS, Ctrl NCoR1kd and DSS NCoR1kd relative to average control values of Ctrl in colon tissues. GAPDH was used for normalisation. f) qRT-PCR analysis of relative fold expression of NCoR1 gene in DSS, Ctrl NCoR1kd and DSS NCoR1kd relative to average values of Ctrl in spleen tissues. GAPDH was used for normalisation g) Representative alcian blue staining in colon tissue sections of Ctrl, DSS, Ctrl NCoR1kd and DSS NCoR1kd mice (n=4 per group) (scale bar=50um). Zoom in images are shown in the inset of the indicated area. h) graph showing goblet cell count in the crypts of Ctrl, DSS, Ctrl NCoR1kd and DSS NCoR1kd mice colon (n=120). Each dot represents (c,d,e,f) one mouse. All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Knockdown, Control, Injection, Quantitative RT-PCR, Expressing, Staining, Cell Characterization

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) Immunoblotting of NCoR1 in HT29 MTX cells upon vitamin D treatment at a concentration of 25uM for 24 hours. The graph on right represents densitometric analysis showing fold intensity of NCoR1 expression calculated by normalising to loading control (Actin) b) Immunoblotting of NCoR1 in HT29 MTX cells upon Tricostatin A treatment at a concentration of 5uM for 24 hours. The graph on right represents densitometric analysis showing fold intensity of NCoR1 expression calculated by normalising to loading control (Actin) c) Graph showing spleen to body weight ratio in Ctrl, DSS, Ctrl VD or DSS VD mice. Each dot represents (a and b) individual experiment and (c) individual mice. All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Western Blot, Concentration Assay, Expressing, Control

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) Schematic representation of the experimental plan for vitamin D-mediated overexpression of NCoR1 in C57Bl/6 mice w/c or w/o 2% DSS in drinking water. Vitamin D was mixed in feed at a dosage of 3000 IU/kg/day with 1% CaCl 2 throughout experiment. Duration of administration of DSS (red), and vitamin D (green triangle) are indicated by respective arrows (Created with BioRender.com ) b) Graph showing the percentage body weight change in Ctrl, DSS, Ctrl VD and DSS VD mice groups. c) Gross morphology of colon and caeca of Ctrl, DSS, Ctrl VD and DSS VD mice. Graph on the right showing colon length quantification. d) Immunoblot of NCoR1 protein in whole tissue lysate of Ctrl, DSS, Ctrl VD and DSS VD mice. The graph on the right represents densitometric analysis showing fold intensity of NCoR1 expression calculated by normalising to loading control (Tubulin) e) Immunoblot of NCoR1 protein in crypt lysate of Ctrl, DSS, Ctrl VD and DSS VD mice. The graph on the right represents densitometric analysis showing fold intensity of NCoR1 expression calculated by normalising to loading control (Tubulin). f) Representative immunohistochemistry (IHC-Alcian blue) images of colon from Ctrl, DSS, Ctrl VD and DSS VD mice stained for NCoR1 (n=4 per group) (scale bar=100um). g) Graph showing goblet cell count in the crypts of Ctrl, DSS, Ctrl VD and DSS VD mice (n=120 crypts per group). Each dot represents (c,d,e) one mouse. All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. See also fig S5a and fig S5c.

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Over Expression, Western Blot, Expressing, Control, Immunohistochemistry, Staining, Cell Characterization

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) Volcano plot of DEGs representing NCoR1 targets. The pink dots represent genes differentially expressed (adjusted P<0.05) in Tricostatin A-treated and control HT29 MTX cells. The symbol-marked dots indicate the genes with the largest negative or positive standardized mean difference. b) Heat map of genes regulated by NCoR1 in Tricostatin A-treated and control HT29 MTX cells represented in colour contrast. Expression of targets is depicted in the gradient of blue to red, indicating lowest to highest expression, respectively. c) Graph showing chromatin immunoprecipitation of KLF16 binding via chromatin pulled by IgG, H3 and NCoR1 antibodies in Tricostain A-treated and control HT29 MTX cells. Quantification done using the fold-over IgG method. d) Immunoblot of NCoR1 and KLF16 in HT29 MTX cells upon transient overexpression of NCoR1(NCoR1 CTL and NCoR1 Up ). The graph on the right represents densitometric analysis showing fold intensity of NCoR1 and KLF16 expression calculated by normalising to loading control (Actin). e) Immunoblot of NCoR1 and KLF16 in HT29 MTX cells upon transient knockdown of NCoR1(siCTL and NCoR1 KD ). The graph on the right represents densitometric analysis showing fold intensity of NCoR1 and KLF16 expression calculated by normalising to loading control (Actin). f) Representative confocal imaging of NCoR1 (blue) stained using anti- NCoR1 antibody and KLF16 (green) stained using anti-KLF16 antibody in HT29 MTX scr and HT29 MTX NCoR1kd cells scale bar=3um. Graphs on the right show fluorescence intensity of NCoR1 and KLF16 measured (n=60). Each dot represents an individual experiment (c, d, and e). All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Control, Expressing, Chromatin Immunoprecipitation, Binding Assay, Western Blot, Over Expression, Knockdown, Imaging, Staining, Fluorescence

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet:

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques:

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) Graph showing binding of AGAP3, KLF16, GLTPD2 and EXOSC6 in chromatin immunoprecipitation (ChIP) using anti- NCoR1 and anti-IgG antibodies in HT29 Undiff , HT29 Diff cells. quantification done using fold over IgG method. b) qRT-PCR analysis of relative fold expression of NCoR1, AGAP3, GLTPD3, EXOSC6 and KLF16 genes upon transient knockdown of NCoR1 with respect to control HT29 MTX cells. GAPDH used for normalization. c) qRT-PCR analysis of relative fold expression of NCoR1, AGAP3, GLTPD3, EXOSC6 and KLF16 genes upon transient overexpression of NCoR1 with respect to control HT29 MTX cells. GAPDH used for normalization. d) Immunoblot of NCoR1 and KLF16 in Nuclear and cytoplasmic fractions of HT29 MTX scr and HT29 MTX NCoR1kd cells. Graph on the right represents densitometric analysis showing fold intensity of NCoR1 and KLF16 expression in the nuclear fraction, calculated by normalizing to loading control (H3 for nuclear fraction). All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Expressing, Knockdown, Control, Over Expression, Western Blot

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) Immunoblotting of NCoR1 and KLF16 in HT29 MTX cells upon transient knockdown of KLF16 (siCTL and siKLF16). The graph on the right represents densitometric analysis showing fold intensity of NCoR1 and KLF16 expression calculated by normalising to loading control (Actin). b) qRT-PCR analysis of relative fold expression of NCoR1, KLF16 and MUC2 genes upon transient knockdown of KLF16 (siKLF16) relative to siCTL in HT29 MTX cells. GAPDH was used for normalisation. c) Representative confocal imaging of MUC2 (green) stained using anti-MUC2 antibody in siCTL and siKLF16 in HT29MTX cells (n=90). Scale bar=2 um. The graph shows MUC2 fluorescence intensity measured. d) JASPAR predicted sequence for KLF16 binding. Image on the right indicates pSCAN predicted site for KLF16 binding on MUC2 promoter. Image Created with BioRender.com . e) Immunoblot of KLF16 in J774, HT29 Undiff ,HT29 Diff , HCT8 and HT29-MTX cell lines. The on the right graph represents NCoR1 expression calculated by normalising to loading control (Actin) specific to each cell type. Each dot represents (a,b and e) individual experiment. All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Western Blot, Knockdown, Expressing, Control, Quantitative RT-PCR, Imaging, Staining, Fluorescence, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) Representative Immunoblot of KLF16 protein in human ulcerative colitis (UC) (n = 8) and control (n = 6) biopsy samples. Graph on the right represents densitometric analysis showing fold intensity of KLF16 expression calculated by normalising to loading control (Tubulin). b) qRT-PCR analysis of relative fold expression of KLF16 gene in human UC patient colonic biopsies (n = 24) relative to average control values (n = 23). 18s was used for normalisation. c) Immunoblot of KLF16 protein in crypt lysate of DSS dynamics Control, DSS1, DSS3 and DSS5 mice. The graph on right represents densitometric analysis showing fold intensity of KLF16 expression calculated by normalising to loading control (Actin). d) Immunoblot of KLF16 protein in whole tissue lysate of NCoR1 knockdown mice i.e. Ctrl, Ctrl NCoR1kd ,DSS and DSS NCoR1kd mice. The graph on right represents densitometric analysis showing fold intensity of KLF16 expression calculated by normalising to loading control (Tubulin). e) Immunoblot of KLF16 protein in whole tissue lysate from the colon of vitamin D-mediated rescue in mice i.e. Ctrl, DSS, Ctrl VD or DSS VD . The graph on right represents densitometric analysis showing fold intensity of KLF16 expression calculated by normalising to loading control (Tubulin). f) Immunoblot of KLF16 protein in crypt lysate from colon of vitamin D mediated rescue in mice i.e. Ctrl, DSS, Ctrl VD or DSS VD . The graph on right represents densitometric analysis showing fold intensity of KLF16 expression calculated by normalising to loading control (Tubulin). Each dot represents (a and b) individual human and (c,d,e and f) individual mice. All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Western Blot, Control, Expressing, Quantitative RT-PCR, Knockdown

Journal: bioRxiv

Article Title: Corepressor NCoR1-mediated regulation of mucin dynamics governs gut inflammation

doi: 10.64898/2026.05.02.722388

Figure Lengend Snippet: a) Immunoblot of NCoR1, KLF16 and mTOR pathway proteins pmTOR, mTOR, pS6, ppS6, p4EBP1, KLF16 and NCoR1 in the cell lysates of HT29 MTX NCoR1kd andHT29 MTX scr cells. Graph below represents densitometric analysis showing fold intensity of respective protein expression calculated by normalising to loading control (Actin). b) Immunoblot of NCoR1, KLF16 and mTOR pathway proteins ppS6 and p4EBP1in the cell lysates of siCTL and siKLF16 HT29 MTX cells. Graph represents densitometric analysis showing fold intensity of respective protein expression of NCoR1, KLF16, ppS6, mTOR, and p4EBP1, calculated by normalizing to loading control (Actin). Each dot represents (a and b) an individual experiment. All data were expressed as means ± SEM, unpaired Student t-test was used to calculate statistical significance * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: A customised, morpholino based NCoR1 specific knockdown model was developed (Gene Tools LLC, USA).

Techniques: Western Blot, Expressing, Control

Journal: Redox Biology

Article Title: Methylation reader MBD2-mediated GPX4 transcriptional repression drives ovarian granulosa cell ferroptosis in PCOS

doi: 10.1016/j.redox.2026.104034

Figure Lengend Snippet: GPX4 suppression is regulated by a repressive complex containing MBD2, MAZ, HDAC3 and NCoR. (a) Peak plot showing the ATAC-seq peak at the Gpx4 locus (Chr10: 80051488–80056439) in ovarian tissues from control (Ctrl, blue) and DHEA-treated (DHEA, red) mice. Orange boxes and asterisks denote regions with increased chromatin accessibility. (b) A heatmap displays the top six transcription factors (TFs) binding to the Gpx4 promoter region in the ATAC-seq analysis, along with the mRNA expression identified by RNA-seq analysis, and the predicted TF motifs and E-values are shown on the right. (c) Schematic representation of the Gpx4 promoter region showing the MAZ binding motif relative to the transcription start site (TSS). (Below) MAZ binding footprint enrichment at the Gpx4 locus in Ctrl (blue) and DHEA-treated (red) mice. Primary ovarian granulosa cells (GCs) were treated with 50 μM DHEA for 48 h in vitro to establish the PCOS model. (d) Western blot analysis of MAZ, NCoR and HDAC3 protein expression in DHEA-treated GCs. GAPDH served as a loading control. Blots are representative of one sample per group. Quantification was presented as means ± SEM, n = 3. ∗ P < 0.05, Student's t-test. (e) Co-immunoprecipitation (Co-IP) assay. Cell lysates were immunoprecipitated (IP) with isoform-matched immunoglobulin (Ig) or antibodies (IP Ab) to MBD2, MAZ, HDAC3, or NCoR, and then immunoprecipitants were assessed for MBD2, MAZ, HDAC3, or NCoR by western blotting reciprocally (the upper panel). The non-IP lysates (Input) were assayed for GAPDH as input controls. (f) Immunofluorescence co-staining was used to determine the expression and localization of MAZ (green), NCoR (red), and HDAC3 (magenta) within GCs. (g) Quantification of protein co-localization from the magnified region in ( f ). (h) Chromatin immunoprecipitation (ChIP) assay. DHEA-treated GCs were in presence or absence of KCC-07 (KCC, 10 μM, 48 h), and the cell lysates were immunoprecipitated with isoform-matched immunoglobulin or antibodies to MBD2, MAZ, NCoR, HDAC3, or pan-acetylated lysine (Pan-Ace), respectively. The genomic DNA (Input) and the antibody-bound DNAs were PCR-amplified with primers covering the MAZ motif on Gpx4 promoter. The PCR products of representative sample per group were analyzed on 1.5 % agarose gels. Quantitative analysis was shown on the right. Data were presented as mean ± SEM, n = 4. ∗ P < 0.05, one-way ANOVA. (i) Western blot analysis. (Left) HDAC3 and GPX4 protein expression in DHEA-treated GCs in the presence or absence of the HDAC3 inhibitor RGFP966 (RGFP, 10 μM, 48 h). (Middle) MAZ and GPX4 protein expression in GCs transfected with negative- (si-Ctrl) or MAZ-targeting (si-MAZ) siRNA, followed by treatment with or without DHEA. (Right) NCoR and GPX4 protein expression in GCs transfected with negative- (si-Ctrl) or NCoR-targeting (si-NCoR) siRNA, followed by DHEA treatment. GAPDH was as a loading control. (j) Quantifications of ( i ). Data were presented as mean ± SEM, n = 3. ∗ P < 0.05, one-way ANOVA. (k) Schematic model of Gpx4 transcriptional repression. A transcriptional repressive complex orchestrated by MBD2, MAZ, HDAC3, and NCoR binds to the hypermethylated Gpx4 promoter, leading to transcriptional suppression.

Article Snippet: Antibodies against MBD2 ( AB188474 , ABclonal), MAZ (21068-1-AP, Proteintech, USA), NCoR (20018-1-AP, Proteintech, USA), HDAC3 (A19537, ABclonal, China), and Pan-Ace (HY– P80179 , MCE) were used for immunoprecipitation.

Techniques: Control, Binding Assay, Expressing, RNA Sequencing, In Vitro, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Immunofluorescence, Staining, Chromatin Immunoprecipitation, Amplification, Transfection

Journal: Redox Biology

Article Title: Methylation reader MBD2-mediated GPX4 transcriptional repression drives ovarian granulosa cell ferroptosis in PCOS

doi: 10.1016/j.redox.2026.104034

Figure Lengend Snippet: Granulosa GPX4 deletion blocks the anti-ferroptotic and ovary-protective effects of MBD2 inhibition in PCOS mice. Gpx4 fl/fl and Gpx4 GC−/− mice were grouped into oil vehicle control (Ctrl), DHEA (60 mg/kg, 21 days)-treated (DHEA), and DHEA-treated with KCC-07 (KCC, 10 mg/kg) treatment (KCC/DHEA) mice ( n = 6). (a) Representative photomicrographs of ovarian sections. Ovarian sections were stained with hematoxylin-eosin (HE; upper panels), Masson trichrome (middle panels), and TUNEL assay (lower panels). Asterisks indicate corpora lutea; black arrows indicate preantral follicles; yellow arrows indicate collagen deposits; white arrows indicate TUNEL-positive cells. (b) Quantification of ( a ). Box-and-whisker plots with data points ( n = 6). ∗ P < 0.05, two-way ANOVA. (c) Western blot analysis of GPX4, 4-HNE, Collagen I (Col1α) and α-SMA protein expression in ovarian tissues. GAPDH served as a loading control. Blots are representative of two samples per group. (d) Quantification of ( c ). Data were presented as mean ± SEM, n = 6. ∗ P < 0.05, two -way ANOVA. (e) A schematic diagram of sequential MBD2 elevation, formation of a transcriptional repressive complex with MAZ, NCoR and HDAC3, binding to the DNMT-hypermethylated Gpx4 promoter, suppression of Gpx4 transcription, and granulosa cell ferroptosis that promotes polycystic ovary syndrome (PCOS) (dashed lines). Conversely, MBD2 inhibition with KCC-07 blocks GPX4 suppression and ferroptotic PCOS (solid lines).

Article Snippet: Antibodies against MBD2 ( AB188474 , ABclonal), MAZ (21068-1-AP, Proteintech, USA), NCoR (20018-1-AP, Proteintech, USA), HDAC3 (A19537, ABclonal, China), and Pan-Ace (HY– P80179 , MCE) were used for immunoprecipitation.

Techniques: Inhibition, Control, Staining, TUNEL Assay, Whisker Assay, Western Blot, Expressing, Binding Assay

Journal: Redox Biology

Article Title: Methylation reader MBD2-mediated GPX4 transcriptional repression drives ovarian granulosa cell ferroptosis in PCOS

doi: 10.1016/j.redox.2026.104034

Figure Lengend Snippet: GPX4 suppression is regulated by a repressive complex containing MBD2, MAZ, HDAC3 and NCoR. (a) Peak plot showing the ATAC-seq peak at the Gpx4 locus (Chr10: 80051488–80056439) in ovarian tissues from control (Ctrl, blue) and DHEA-treated (DHEA, red) mice. Orange boxes and asterisks denote regions with increased chromatin accessibility. (b) A heatmap displays the top six transcription factors (TFs) binding to the Gpx4 promoter region in the ATAC-seq analysis, along with the mRNA expression identified by RNA-seq analysis, and the predicted TF motifs and E-values are shown on the right. (c) Schematic representation of the Gpx4 promoter region showing the MAZ binding motif relative to the transcription start site (TSS). (Below) MAZ binding footprint enrichment at the Gpx4 locus in Ctrl (blue) and DHEA-treated (red) mice. Primary ovarian granulosa cells (GCs) were treated with 50 μM DHEA for 48 h in vitro to establish the PCOS model. (d) Western blot analysis of MAZ, NCoR and HDAC3 protein expression in DHEA-treated GCs. GAPDH served as a loading control. Blots are representative of one sample per group. Quantification was presented as means ± SEM, n = 3. ∗ P < 0.05, Student's t-test. (e) Co-immunoprecipitation (Co-IP) assay. Cell lysates were immunoprecipitated (IP) with isoform-matched immunoglobulin (Ig) or antibodies (IP Ab) to MBD2, MAZ, HDAC3, or NCoR, and then immunoprecipitants were assessed for MBD2, MAZ, HDAC3, or NCoR by western blotting reciprocally (the upper panel). The non-IP lysates (Input) were assayed for GAPDH as input controls. (f) Immunofluorescence co-staining was used to determine the expression and localization of MAZ (green), NCoR (red), and HDAC3 (magenta) within GCs. (g) Quantification of protein co-localization from the magnified region in ( f ). (h) Chromatin immunoprecipitation (ChIP) assay. DHEA-treated GCs were in presence or absence of KCC-07 (KCC, 10 μM, 48 h), and the cell lysates were immunoprecipitated with isoform-matched immunoglobulin or antibodies to MBD2, MAZ, NCoR, HDAC3, or pan-acetylated lysine (Pan-Ace), respectively. The genomic DNA (Input) and the antibody-bound DNAs were PCR-amplified with primers covering the MAZ motif on Gpx4 promoter. The PCR products of representative sample per group were analyzed on 1.5 % agarose gels. Quantitative analysis was shown on the right. Data were presented as mean ± SEM, n = 4. ∗ P < 0.05, one-way ANOVA. (i) Western blot analysis. (Left) HDAC3 and GPX4 protein expression in DHEA-treated GCs in the presence or absence of the HDAC3 inhibitor RGFP966 (RGFP, 10 μM, 48 h). (Middle) MAZ and GPX4 protein expression in GCs transfected with negative- (si-Ctrl) or MAZ-targeting (si-MAZ) siRNA, followed by treatment with or without DHEA. (Right) NCoR and GPX4 protein expression in GCs transfected with negative- (si-Ctrl) or NCoR-targeting (si-NCoR) siRNA, followed by DHEA treatment. GAPDH was as a loading control. (j) Quantifications of ( i ). Data were presented as mean ± SEM, n = 3. ∗ P < 0.05, one-way ANOVA. (k) Schematic model of Gpx4 transcriptional repression. A transcriptional repressive complex orchestrated by MBD2, MAZ, HDAC3, and NCoR binds to the hypermethylated Gpx4 promoter, leading to transcriptional suppression.

Article Snippet: The lysates of ovaries were immunoprecipitated with antibodies to MBD2 (ab188474, Abcam, UK), MAZ (21068-1-AP, Proteintech, USA), HDAC3 (A19537, ABclonal, China), NCoR (20018-1-AP, Proteintech, USA), or an isotype-matched immunoglobulin (Ig) followed by Protein A/G Magnetic Beads (PB101, Vazyme, China), and then immunoprecipitants were assayed by Western blot with antibodies to MBD2, MAZ, HDAC3 or NCoR, respectively.

Techniques: Control, Binding Assay, Expressing, RNA Sequencing, In Vitro, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Immunofluorescence, Staining, Chromatin Immunoprecipitation, Amplification, Transfection