kcc 07  (MedChemExpress)

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: A PCOS cell model was established by DHEA (50 μM, 48 h) treatment, followed by KCC-07 (10 μM, 48 h), 5-Aza (10 μM, 24 h), RGFP966 (10 μM, 48 h, HY-13909, MCE, USA), and/or Liproxstatin-1 (200 nM, 48 h, HY-12726, MCE, USA) treatments.

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: MBD2 inhibition by KCC-07 alleviates lipid peroxidation in granulosa cells. Primary granulosa cells (GCs) treated with DHEA (50 μM) in presence or absence with KCC-07 (KCC, 10 μM), or with Liproxstatin-1 (Lip-1, 200 nM), or with 5-Azacytidine (5-Aza/5Az, 10 μM, 24 h), respectively. (a) Representative micrographs of C11-BODIPY assay (upper three panels) and TUNEL staining (below panel). (b) Quantification of ( a ). Data were presented as Box-and-whisker plots with data points ( n = 4). ∗ P < 0.05, two-way ANOVA. (c) Western blot analysis of 4-HNE, GPX4, FSHR, and Cyp19a1 protein expression in GCs. GAPDH was as a loading control. (d) Quantification of ( c ). Data were presented as mean ± SEM, n = 3. ∗ P < 0.05, one-way ANOVA.

Article Snippet: A PCOS cell model was established by DHEA (50 μM, 48 h) treatment, followed by KCC-07 (10 μM, 48 h), 5-Aza (10 μM, 24 h), RGFP966 (10 μM, 48 h, HY-13909, MCE, USA), and/or Liproxstatin-1 (200 nM, 48 h, HY-12726, MCE, USA) treatments.

Techniques: Inhibition, TUNEL Assay, Staining, Whisker Assay, 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: Pharmacological inhibition of DNMT and MBD2 alleviates ovarian ferroptosis and PCOS pathologies. The oil vehicle control (Ctrl) or DHEA (60 mg/kg, 21 days)-treated mice were treated with or without KCC-07 (KCC, 10 mg/kg) or 5-Azacytidine (5-Aza/5Az, 1.5 mg/kg), respectively ( n = 10). (a) Representative micrographs of ovarian sections. Ovarian sections were stained with hematoxylin-eosin (HE; left panels), Masson trichrome (middle panels), TUNEL assay (second panels from right), and by immunohistochemistry (IHC; right panels) for GPX4 staining. Asterisks indicate corpora lutea; black arrows indicate preantral follicles; yellow arrows indicate collagen deposits; white arrows indicate TUNEL-positive cells; red arrows indicate IHC-positive cells. (b) Quantification of ( a ) and malondialdehyde (MDA) levels in ovarian tissues. Data were presented as Box-and-whisker plots with data points ( n = 6). ∗ P < 0.05, two-way ANOVA. (c) Representative transmission electron microscopy (TEM) images of ovarian tissues. Yellow arrows indicate mitochondria with morphological changes consistent with ferroptosis. (d) Quantification of ( c ). Box-and-whisker plots with data points ( n = 6). ∗ P < 0.05, two-way ANOVA. (e) Western blot analysis of GPX4, 4-HNE, α-SMA, and Collagen I (Col1α) protein expression in ovarian tissues. GAPDH served as a loading control. Blots are representative of two samples per group. (f) Quantification of ( e ). Data were presented as mean ± SEM, n = 6. ∗ P < 0.05, one-way ANOVA.

Article Snippet: A PCOS cell model was established by DHEA (50 μM, 48 h) treatment, followed by KCC-07 (10 μM, 48 h), 5-Aza (10 μM, 24 h), RGFP966 (10 μM, 48 h, HY-13909, MCE, USA), and/or Liproxstatin-1 (200 nM, 48 h, HY-12726, MCE, USA) treatments.

Techniques: Inhibition, Control, Staining, TUNEL Assay, Immunohistochemistry, Whisker Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing

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 inhibition by RSL3 abrogates the anti-ferroptosis effects of KCC-07 in PCOS mice. Control and RSL3 (5 mg/kg, the last 10 days)-treated 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 = 10). (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.

Article Snippet: A PCOS cell model was established by DHEA (50 μM, 48 h) treatment, followed by KCC-07 (10 μM, 48 h), 5-Aza (10 μM, 24 h), RGFP966 (10 μM, 48 h, HY-13909, MCE, USA), and/or Liproxstatin-1 (200 nM, 48 h, HY-12726, MCE, USA) treatments.

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

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: A PCOS cell model was established by DHEA (50 μM, 48 h) treatment, followed by KCC-07 (10 μM, 48 h), 5-Aza (10 μM, 24 h), RGFP966 (10 μM, 48 h, HY-13909, MCE, USA), and/or Liproxstatin-1 (200 nM, 48 h, HY-12726, MCE, USA) treatments.

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