Journal: iScience

Article Title: Bazedoxifene rescues hepatocytes from chemically induced oxidative ferroptotic injury in vivo and in vitro by inhibiting protein disulfide isomerase

doi: 10.1016/j.isci.2026.114931

Figure Lengend Snippet: Role of ferroptosis in APAP-induced liver injury in C57BL/6J mice (A) IHC staining of GPX4 (upper panel) and PTGS2 (middle panel) proteins and TUNEL staining (bottom panel) in hepatocytes of C57BL/6J mice treated with APAP ± BAZ at indicated doses for 24 h. For each staining, the upper images represent the original low-magnification views, and the lower images show the corresponding enlarged areas. Scale bars = 1 mm (upper images) and 250 μm (lower images). (B–D) The respective quantitative intensity values of GPX4 protein level (B, n = 3), PTGS2 protein level (C, n = 3), and TUNEL staining level (D, n = 3). All quantitative data are presented as mean ± S.D. (∗∗ or ## p < 0.01).

Article Snippet: For detection of GPX4 and COX-2, paraffin-embedded liver sections were incubated overnight at 4°C with either anti-GPX4 antibody (#HY- P80450 , 1:500; MedChemExpress, Monmouth Junction, NJ, USA) or anti-COX-2 antibody (#12282S, 1:1000; Cell Signaling Technology, Boston, MA, USA).

Techniques: Immunohistochemistry, TUNEL Assay, Staining

Journal: BMC Biology

Article Title: Hexadecanamide alleviates experimental colitis in mice and modifies the gut microbiome

doi: 10.1186/s12915-026-02530-w

Figure Lengend Snippet: HEX inhibits DSS-induced ferroptosis. A - C Concentration of Fe. 2+ ( A ), MDA ( B ) and GSH ( C ) in the colon from each group ( n = 5). D , E The relative mRNA levels of PTGS2 ( D ) and GPX4 ( E ) were determined using qPCR. F Representative western blot images of colon PTGS2, GPX4, ACSL4 and SLC7A11 from different groups. G. Representative colonic H&E-stained sections from the indicated mice (scale bar: 50 μm). H Histological scores of the colon from different groups. I - K The levels of inflammatory parameters, including MPO activity ( I ), TNF-α ( J ), and IL-1β ( K ) were detected. L , M The relative mRNA levels of ferroptosis-associated genes from the indicated groups were detected by qPCR. N The relative intensities of PTGS2, GPX4, ACSL4 and SLC7A11 were measured. O Representative protein levels of SQSTM1 and LC3 in the colon of mice from each group. P Representative western blot images of NCOA4 and FTH proteins in the colon tissues from the indicated groups. Data are presented as mean ± SD followed by Tukey’s test (A-F and H-P). ns, no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001 suggest significant statistical difference by one-way ANOVA (A-F and H-P)

Article Snippet: After blocking with 5% skim milk for 3 h at room temperature, the membranes were incubated with specific antibodies, including β-actin (1:1000; #AF7018, Affinity Biosciences, OH, USA), ZO-1 (1:1000; #AF5145, Affinity Biosciences, OH, USA), Occludin (1:1000; #DF7504, Affinity Biosciences, OH, USA), Claudin-3 (1:1000; #AF0129, Affinity Biosciences, OH, USA), PTGS2 (1:1000; #AF7003, Affinity Biosciences, OH, USA), GPX4 (1:1000; bs-3884R, Bioss, Beijing, China), NCOA4 (1:1000; YT0302, Immunoway DE, USA), FTH1 (1:1000; bs-5907R, Bioss, Beijing, China), LC3 (1:1000; #AF5402, Affinity Biosciences, OH, USA), SQSTM1 (1:1000; #AF5384, Affinity Biosciences, OH, USA), SLC7A11 (1:1000; #DF12509, Affinity Biosciences, OH, USA), ACSL4 (1:1000; #DF12141, Affinity Biosciences, OH, USA) were used to measure target proteins.

Techniques: Concentration Assay, Western Blot, Staining, Activity Assay

Journal: BMC Biology

Article Title: Hexadecanamide alleviates experimental colitis in mice and modifies the gut microbiome

doi: 10.1186/s12915-026-02530-w

Figure Lengend Snippet: HEX-FMT + DSS treatment alleviates ferroptosis and colonic damage better than Ctrl-FMT + DSS treatment. A - C Levels of Fe 2+ ( A ), MDA ( B ) and GSH ( C ) in the colon ( n = 5). D , E The relative mRNA expression levels of PTGS2 ( D ) and GPX4 ( E ) were measured by qPCR. F Representative western blot images of PTGS2, GPX4, ACSL4, and SLC7A11 proteins in the colon from the indicated mice. G Representative images of SQSTM1 and LC3 from the differently treated mice. H Representative images of FTH and NCOA4 were shown by western boltting. I Representative western blot images of TJ proteins in the colon tissues from the indicated groups. J , K The alcian blue (AB) staining was performed to examine the mucus layer of the colon from different mice, and the mucin-2 level was assessed by immunohistochemistry (scale bars, 50 μm). L The mucin-2 positive-stained cells were shown in brown. Student’s t test (A-I and L) was performed, and values are presented as the mean ± SD (A-I and L). * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate significant difference from each group. ns, no significance

Article Snippet: After blocking with 5% skim milk for 3 h at room temperature, the membranes were incubated with specific antibodies, including β-actin (1:1000; #AF7018, Affinity Biosciences, OH, USA), ZO-1 (1:1000; #AF5145, Affinity Biosciences, OH, USA), Occludin (1:1000; #DF7504, Affinity Biosciences, OH, USA), Claudin-3 (1:1000; #AF0129, Affinity Biosciences, OH, USA), PTGS2 (1:1000; #AF7003, Affinity Biosciences, OH, USA), GPX4 (1:1000; bs-3884R, Bioss, Beijing, China), NCOA4 (1:1000; YT0302, Immunoway DE, USA), FTH1 (1:1000; bs-5907R, Bioss, Beijing, China), LC3 (1:1000; #AF5402, Affinity Biosciences, OH, USA), SQSTM1 (1:1000; #AF5384, Affinity Biosciences, OH, USA), SLC7A11 (1:1000; #DF12509, Affinity Biosciences, OH, USA), ACSL4 (1:1000; #DF12141, Affinity Biosciences, OH, USA) were used to measure target proteins.

Techniques: Expressing, Western Blot, Staining, Immunohistochemistry

Journal: iScience

Article Title: Bazedoxifene rescues hepatocytes from chemically induced oxidative ferroptotic injury in vivo and in vitro by inhibiting protein disulfide isomerase

doi: 10.1016/j.isci.2026.114931

Figure Lengend Snippet: Role of ferroptosis in APAP-induced liver injury in C57BL/6J mice (A) IHC staining of GPX4 (upper panel) and PTGS2 (middle panel) proteins and TUNEL staining (bottom panel) in hepatocytes of C57BL/6J mice treated with APAP ± BAZ at indicated doses for 24 h. For each staining, the upper images represent the original low-magnification views, and the lower images show the corresponding enlarged areas. Scale bars = 1 mm (upper images) and 250 μm (lower images). (B–D) The respective quantitative intensity values of GPX4 protein level (B, n = 3), PTGS2 protein level (C, n = 3), and TUNEL staining level (D, n = 3). All quantitative data are presented as mean ± S.D. (∗∗ or ## p < 0.01).

Article Snippet: GPX4 Antibody (YA399) , MedChemExpress , Cat# P80450 ; RRID: AB_3102526.

Techniques: Immunohistochemistry, TUNEL Assay, Staining

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: The PCOS ovary exhibits severer ferroptosis and transcriptional suppression of GPX4. PCOS mouse model was constructed by subcutaneous injection of dehydroepiandrosterone (DHEA, 60 mg/kg) for 21 consecutive days, while control (Ctrl) mice were injected with oil vehicle ( n = 10 in each group). (a) Body weight (g) and ovary weight (mg) of control (Ctrl) and DHEA-treated (DHEA) mice. ∗ P < 0.05, Student's t -test. (b) Representative H&E-stained ovarian sections from Ctrl and DHEA mice. Asterisks indicate corpora lutea; black arrows indicate preantral follicles. (c) Quantification of ( b ). Box-and-whisker plots with data points ( n = 6). ∗ P < 0.05, Student's t -test. (d) Representative photomicrographs of ovarian sections from Ctrl and DHEA mice following Masson trichrome (collagen deposition, yellow arrows), TUNEL (positive cells, white arrows), and Perl's (iron deposition, red arrows) staining, alongside a transmission electron microscopy (TEM) micrograph (ferroptotic mitochondria, orange arrows). (e) Quantification of ( d ). Box-and-whisker plots with data points ( n = 6). ∗ P < 0.05, Student's t -test. (f) Quantitative real-time PCR (qRT-PCR) analysis of ovarian Gpx4 mRNA in Ctrl and DHEA mice. The Beta-actin gene ( Actb ) was used as an internal control. Data were presented as mean ± SEM, n = 6. ∗ P < 0.05, Student's t -test. (g) Western blot analysis of ovarian GPX4, 4-HNE, α-SMA, and Collagen I (Col1α) proteins from Ctrl and DHEA mice. GAPDH served as a loading control. Blots (left panel) are representative of two samples per group. Quantitative data (right panel) were presented as mean ± SEM, n = 6. ∗ P < 0.05, Student's t -test. (h) Volcano plot of gene expression profile from the database [GEO Dataset Accession Number: GSE293353 and GSE277906 ], comprising RNA-seq data ovarian granulosa cells of 24 women diagnosed with PCOS and 24 healthy control women. The number and position of genes statistic-significantly increased (red, 262), no difference (gray, 20,075), or decreased (blue, 606) including GPX4 (Log 2 (fold-change) = −0.55294), orange point was marked. (i) Representative photomicrographs of ovarian sections from Ctrl and DHEA mice stained for GPX4 by immunohistochemistry (IHC) staining. (j) Western blot analysis of 4-HNE and GPX4 expression in primary granulosa cells (GCs) treated with or without DHEA (50 μM, 48 h). GAPDH was used as a loading control. Quantification was shown below. Data were presented as mean ± SEM, n = 4. ∗ P < 0.05, Student's t -test. (k) Representative images of C11-BODIPY and TUNEL staining in GCs treated with or without DHEA. (Left) Non-oxidized (N-) and oxidized (O-) BODIPY were in red and green respectively, and then merged. (Right) The positively-stained cells by TUNEL (TUNEL Bright Red kit) were shown.

Article Snippet: To further confirm GPX4 as a critical target for MBD2-driven GC ferroptosis, pharmacologic GPX4 inhibition with RSL3 (5 mg/kg, intraperitoneal injection for 10 days; HY-100218A, MCE, USA) or GC-specific Gpx4 deletion mice were used to establish the PCOS model.

Techniques: Construct, Injection, Control, Staining, Whisker Assay, TUNEL Assay, Transmission Assay, Electron Microscopy, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Gene Expression, RNA Sequencing, Immunohistochemistry, 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 knockout in granulosa cells aggregates ovarian ferroptosis. (a) Generation of granulosa cell-specific Gpx4 knockout mice ( Gpx4 GC−/− ) by crossing Gpx4 fl/fl with Cyp19α1 -Cre mice. Gpx4 locus in wild-type mice were represented by boxes (exons 1–7). The positions of Cyp19a1-Cre genotyping PCR primers F2 and R2 (arrows), and the loxP sites (blue triangles) in Gpx4 fl/fl mice and the genotyping PCR primers F1 and R1 (arrows) were depicted. (b) Genotyping of mice by PCR. Agarose gel electrophoresis shows PCR products from WT, Gpx4 fl/- , Gpx4 fl/fl and Gpx4 GC−/− mice. (c) Appearance of Gpx4 fl/fl and Gpx4 GC−/− mice at 7 weeks. Representative images of three mice per group were shown. (d) Appearance of ovaries from Gpx4 fl/fl and Gpx4 GC−/− mice at 7 weeks. Representative images of three pairs of ovaries per group were shown. (e) Quantification of body weight (g) and ovary weight (mg) of Gpx4 fl/fl and Gpx4 GC−/− mice at 7 weeks. Data were presented as mean ± SEM, n = 6. ∗ P < 0.05, Student's t -test. (f) Quantification of malondialdehyde (MDA) levels in ovarian tissues. Data were presented as violin plots with data points ( n = 6). ∗ P < 0.05, one-way ANOVA. (g) Representative photomicrographs of ovarian sections. Serial sections were stained with hematoxylin-eosin (HE; left panels), Masson trichrome (middle panels), and TUNEL assay (right panels). Asterisks indicate corpora lutea; black arrows indicate preantral follicles; yellow arrows indicate collagen deposits; white arrows indicate TUNEL-positive cells. (h) Quantification of ( g ). Box-and-whisker plots with data points ( n = 6). ∗ P < 0.05, one-way ANOVA. (i) Western blot analysis of GPX4, 4-HNE, α-SMA, and Collagen I (Col1α) expression in ovarian tissues. GAPDH served as a loading control. Blots are representative of two samples per group. (j) Quantification of ( i ). Data were presented as mean ± SEM, n = 6. ∗ P < 0.05, one-way ANOVA.

Article Snippet: To further confirm GPX4 as a critical target for MBD2-driven GC ferroptosis, pharmacologic GPX4 inhibition with RSL3 (5 mg/kg, intraperitoneal injection for 10 days; HY-100218A, MCE, USA) or GC-specific Gpx4 deletion mice were used to establish the PCOS model.

Techniques: Knock-Out, Agarose Gel Electrophoresis, Staining, TUNEL Assay, 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: GPX4 suppression is triggered by DNMT and MBD2-mediated Gpx4 promoter hypermethylation. (a) Schematic diagrams of the mouse Gpx4 promoter. The positions of the CpG island (gray area) and the methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) primers (boxes) are shown. (b) MSP analysis of the Gpx4 promoter in control (Ctrl) and DHEA-treated (DHEA) mice. (Left) Agarose gel images of MSP products from two randomly selected samples per group ( n = 6); (Right) Quantification of the methylated-to-unmethylated ratio, normalized to input. Data were presented as mean ± SEM, n = 6. ∗ P < 0.05, Student's t -test. (c) BSP analysis of the Gpx4 promoter in Ctrl and DHEA mice. Quantification of the percentage of methylated CpG sites was on the right side. Data were presented as Box-and-whisker plots with data points. ∗ P < 0.05, Student's t -test. (d) Heatmap of normalized expression (FPKM) for 23 key transcription regulation-related genes from RNA-seq analysis of Ctrl and DHEA mice. The methylation-related methyltransferases DNMT1 and DNMT3a, significantly increase, and are highlighted in red. (e) Representative photomicrographs of ovarian sections from Ctrl and DHEA mice stained for DNMT1, DNMT3a and MBD2 by immunohistochemistry (IHC) staining. Positively-stained granulosa cells were indicated by arrows. (f) Western blot analysis of DNMT1, DNMT3a, DNMT3b, MBD2, MeCP2, MBD4, MBD3 and MBD1 proteins in ovarian tissues. GAPDH served as a loading control. Blots are representative of two samples per group. Quantification was presented as mean ± SEM, n = 6. ∗ P < 0.05, Student's t -test. (g) MSP analysis of the Gpx4 promoter in ovarian tissues from four treatment groups: Ctrl, 5-Azacytidine (5-Aza, 1.5 mg/kg, the last 10 days), DHEA (60 mg/kg, 21 days), and 5-Aza/DHEA. Agarose gel showing one representative sample per group. Right panel: Quantification of the methylated-to-unmethylated ratio, normalized to input. Data are presented as mean ± SEM, n = 6. ∗ P < 0.05, one-way ANOVA. (h) BSP analysis of the Gpx4 promoter. CpG methylation patterns from three mice per group (M1-M3), with five clones sequenced per mouse. Each row is a clone; each circle is a CpG site (open: unmethylated; closed: methylated). One box encompasses the data from one mouse. Quantification of the percentage of methylated CpG sites was on the right side. Data were presented as Box-and-whisker plots with data points. ∗ P < 0.05, one-way ANOVA.

Article Snippet: To further confirm GPX4 as a critical target for MBD2-driven GC ferroptosis, pharmacologic GPX4 inhibition with RSL3 (5 mg/kg, intraperitoneal injection for 10 days; HY-100218A, MCE, USA) or GC-specific Gpx4 deletion mice were used to establish the PCOS model.

Techniques: Methylation, Methylation Sequencing, Control, Agarose Gel Electrophoresis, Whisker Assay, Expressing, RNA Sequencing, Staining, Immunohistochemistry, Western Blot, CpG Methylation Assay, Clone 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: To further confirm GPX4 as a critical target for MBD2-driven GC ferroptosis, pharmacologic GPX4 inhibition with RSL3 (5 mg/kg, intraperitoneal injection for 10 days; HY-100218A, MCE, USA) or GC-specific Gpx4 deletion mice were used to establish the PCOS model.

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: To further confirm GPX4 as a critical target for MBD2-driven GC ferroptosis, pharmacologic GPX4 inhibition with RSL3 (5 mg/kg, intraperitoneal injection for 10 days; HY-100218A, MCE, USA) or GC-specific Gpx4 deletion mice were used to establish the PCOS model.

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: To further confirm GPX4 as a critical target for MBD2-driven GC ferroptosis, pharmacologic GPX4 inhibition with RSL3 (5 mg/kg, intraperitoneal injection for 10 days; HY-100218A, MCE, USA) or GC-specific Gpx4 deletion mice were used to establish the PCOS model.

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: To further confirm GPX4 as a critical target for MBD2-driven GC ferroptosis, pharmacologic GPX4 inhibition with RSL3 (5 mg/kg, intraperitoneal injection for 10 days; HY-100218A, MCE, USA) or GC-specific Gpx4 deletion mice were used to establish the PCOS model.

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: To further confirm GPX4 as a critical target for MBD2-driven GC ferroptosis, pharmacologic GPX4 inhibition with RSL3 (5 mg/kg, intraperitoneal injection for 10 days; HY-100218A, MCE, USA) or GC-specific Gpx4 deletion mice were used to establish the PCOS model.

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

Journal: Indian Journal of Ophthalmology

Article Title: Ranitidine protects Müller cells against ferroptosis in diabetic retinopathy by regulating the AKT1 / GSK3β pathway

doi: 10.4103/IJO.IJO_1522_25

Figure Lengend Snippet: Ra influences the AKT1 / GSK3β signaling pathway under high-glucose conditions. a and b. Western blot analysis assessed AKT1 and p- AKT1 protein levels in HG-treated rMC-1 cells. a and c. Western blot analysis assessed GSK3β and p- GSK3β protein levels in HG-treated rMC-1 cells. d-f. Effects of Ra on GPX4 and xCT expression in HG-treated rMC-1 cells assessed by Western blotting ( n = 3, *P < 0.05, ns: no significance, compared to HG group). Ra: Ranitidine, HG: High-glucose, rMC-1: Mouse retinal Müller cells, GPX4: Glutathione peroxidase 4, xCT: Cystine/glutamate transporter

Article Snippet: After being closed with 5% bovine serum albumin blocking solution for 2 h at room temperature, the membranes were incubated with primary antibodies against GPX4 (Med Chem Express, USA) (1:1000), xCT (Med Chem Express, USA) (1:1000), GSK3β (Med Chem Express, USA) (1:1000), p- GSK3β (Med Chem Express, USA) (1:1000), AKT1 (Zenbio, USA) (1:1000), or p- AKT1 (Med Chem Express, USA) (1:1000) proteins at 4°C overnight.

Techniques: Western Blot, Expressing

Journal: Indian Journal of Ophthalmology

Article Title: Ranitidine protects Müller cells against ferroptosis in diabetic retinopathy by regulating the AKT1 / GSK3β pathway

doi: 10.4103/IJO.IJO_1522_25

Figure Lengend Snippet: Ra inhibits HG-induced ferroptosis in rMC-1 cells, depending on GSK3β . a-c. The effects of Ra on the expression levels of AKT1 , P- AKT1 , GSK3β , and p- GSK3β in HG-cultured rMC-1 cells were assessed by Western blotting. d-f. The effects of Ra on the expression levels of GPX4 and xCT in HG-cultured rMC-1 cells were assessed by Western blotting ( n = 3, **P < 0.01, ***P < 0.001, ns: no significance, compared to HG group; # P < 0.05, ### P < 0.001, ns: no significance, compared to HG + Ra group). Ra: Ranitidine, HG: High-glucose, rMC-1: Mouse retinal Müller cells, GPX4: Glutathione peroxidase 4, xCT: Cystine/glutamate transporter

Article Snippet: After being closed with 5% bovine serum albumin blocking solution for 2 h at room temperature, the membranes were incubated with primary antibodies against GPX4 (Med Chem Express, USA) (1:1000), xCT (Med Chem Express, USA) (1:1000), GSK3β (Med Chem Express, USA) (1:1000), p- GSK3β (Med Chem Express, USA) (1:1000), AKT1 (Zenbio, USA) (1:1000), or p- AKT1 (Med Chem Express, USA) (1:1000) proteins at 4°C overnight.

Techniques: Expressing, Cell Culture, Western Blot

Journal: Poultry Science

Article Title: Ginsenoside 20(S)-Rg3 alleviates cage stress induced liver injury in laying ducks

doi: 10.1016/j.psj.2025.106254

Figure Lengend Snippet: Rg3 alleviated cage stress induced liver injury by suppressing inflammation and promoting autophagy. (A) Volcano plot (Rg3-H vs. ST); expression of inflammation-related genes in the liver of laying duck; IF of F4/80 protein in the liver of laying ducks. (B-C) Volcano plot (Rg3-H vs. ST); expression of antioxidant-related genes in the liver of laying duck; IF of GPX4 and ACSL4 protein in the liver of laying duck. (D) Volcano plot (Rg3-H vs. ST); expression of autophagy-related genes in the liver of laying duck; IF of LC3B protein in the liver of laying duck. Data are presented as the mean ± SEM; Intergroup differences were marked with lowercase letters. Different letters indicate statistically significant differences between groups (P < 0.05).

Article Snippet: Subsequently, 5 % bovine serum albumin (BSA) was added for blocking at room temperature for 1 h. Primary antibodies against F4/80 (28463-1-AP, Proteintech), GPX4 (30388-1-AP, Proteintech), ACSL4 (sc-365230, Santa Cruz), and LC3B (14600-1-AP, Proteintech) were added, and the sections were incubated overnight at 4°C.

Techniques: Expressing

Journal: Poultry Science

Article Title: Ginsenoside 20(S)-Rg3 alleviates cage stress induced liver injury in laying ducks

doi: 10.1016/j.psj.2025.106254

Figure Lengend Snippet: Rg3 suppressed hepatic ferroptosis by regulating the expression of GPX4 and ACSL4. (A) Volcano plot (Rg3-H vs. ST); expression of ferroptosis-related genes in the liver of laying duck; IF of GPX4 and ACSL4 protein in the liver of laying duck. Data are presented as the mean ± SEM; Intergroup differences were marked with lowercase letters. Different letters indicate statistically significant differences between groups (P < 0.05).

Article Snippet: Subsequently, 5 % bovine serum albumin (BSA) was added for blocking at room temperature for 1 h. Primary antibodies against F4/80 (28463-1-AP, Proteintech), GPX4 (30388-1-AP, Proteintech), ACSL4 (sc-365230, Santa Cruz), and LC3B (14600-1-AP, Proteintech) were added, and the sections were incubated overnight at 4°C.

Techniques: Expressing