Journal: Cells

Article Title: SIRT3 Deficiency Enhances Ferroptosis and Promotes Cardiac Fibrosis via p53 Acetylation

doi: 10.3390/cells12101428

Figure Lengend Snippet: Loss of SIRT3 resulted in cardiac remodeling and ferroptosis. ( A ) Representative images of H&E, Masson’s trichrome and DHE stains of whole heart sections in WT mice and SIRT3KO mice. ( B ) Quantification of cardiomyocyte sizes, interstitial fibrosis area and fluorescence-integrated density of DHE assay in the indicated groups ( n = 3). ( C , D ) Immunoblots and analysis of α-SMA, p53, acetylated p53, GPX-4 and corresponding GAPDH in the indicated mouse hearts ( n = 3–4). Mean ± S.D., ** p < 0.01, *** p < 0.001.

Article Snippet: Equal amounts of protein were run in 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane and then incubated with the primary antibodies at 4 °C overnight: β-myosin heavy chain (β-MHC; 1:1000, Abcam, Cambridge, MA, USA), α-smooth muscle actin (α-SMA; 1:1000, Abcam), 4-hydroxynonenal (4-HNE; 1:1000, Abcam), p53 acetylation (1:1000, Abcam), p53 (1:1000; Cell signaling, Danvers, MA, USA), glutathione peroxidase 4 (GPX-4) (1:1000; Novus Bio, Littleton, CO, USA) and TGF-β1 (1:500; Santa Cruz, CA, USA).

Techniques: Fluorescence, Western Blot

Journal: Cells

Article Title: SIRT3 Deficiency Enhances Ferroptosis and Promotes Cardiac Fibrosis via p53 Acetylation

doi: 10.3390/cells12101428

Figure Lengend Snippet: SIRT3-acetylated p53 mediates ferroptosis in H9c2 myofibroblasts. ( A ) Immunoblots and analysis of p53 acetylation, GPX-4 and GAPDH in H9c2 cells treated with Ad-SIRT3 alone or treated with Ad-SIRT3 and Erastin ( n = 3). ( B ) Immunoblots and analysis of p53 acetylation, GPX-4 and GAPDH in H9c2 cells treated with/without Erastin and C646 ( n = 3). ( C ) Representative images of DHE-stained H9c2 cells treated with/without Erastin and C646. ( D ) Quantification of ROS fluorescence integrated density and ferrous OD value in the indicated H9c2 cells treated with/without Erastin and C646 ( n = 3). Mean ± S.D., * p < 0.05, ** p < 0.01.

Article Snippet: Equal amounts of protein were run in 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane and then incubated with the primary antibodies at 4 °C overnight: β-myosin heavy chain (β-MHC; 1:1000, Abcam, Cambridge, MA, USA), α-smooth muscle actin (α-SMA; 1:1000, Abcam), 4-hydroxynonenal (4-HNE; 1:1000, Abcam), p53 acetylation (1:1000, Abcam), p53 (1:1000; Cell signaling, Danvers, MA, USA), glutathione peroxidase 4 (GPX-4) (1:1000; Novus Bio, Littleton, CO, USA) and TGF-β1 (1:500; Santa Cruz, CA, USA).

Techniques: Western Blot, Staining, Fluorescence

Journal: Cells

Article Title: SIRT3 Deficiency Enhances Ferroptosis and Promotes Cardiac Fibrosis via p53 Acetylation

doi: 10.3390/cells12101428

Figure Lengend Snippet: Inhibition of acetylated p53 rescued ferroptosis and cardiac fibrosis in SIRT3KO mice. ( A ) Representative images of H&E-stained and Masson’s trichrome-stained whole heart sections and quantification of cardiomyocyte sizes and interstitial fibrosis area in WT mice, SIRT3KO mice and SIRT3KO/p534KR mice ( n = 3–4). ( B ) Immunoblots and analysis of α-SMA, p53, p53 acetylation and GAPDH in the indicated mouse hearts ( n = 3–4). ( C ) Representative images of DHE-stained whole heart sections in the indicated mouse hearts. ( D ) Immunoblots and analysis of GPX-4 and GAPDH ratio in the indicated mouse hearts ( n = 3–4). ( E ) Quantification of ferrous iron OD value in the indicated H9c2 cells ( n = 3). Mean ± S.D., * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Equal amounts of protein were run in 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane and then incubated with the primary antibodies at 4 °C overnight: β-myosin heavy chain (β-MHC; 1:1000, Abcam, Cambridge, MA, USA), α-smooth muscle actin (α-SMA; 1:1000, Abcam), 4-hydroxynonenal (4-HNE; 1:1000, Abcam), p53 acetylation (1:1000, Abcam), p53 (1:1000; Cell signaling, Danvers, MA, USA), glutathione peroxidase 4 (GPX-4) (1:1000; Novus Bio, Littleton, CO, USA) and TGF-β1 (1:500; Santa Cruz, CA, USA).

Techniques: Inhibition, Staining, Western Blot

Journal: International Journal of Nanomedicine

Article Title: Anti-Oxidant and Anti-Endothelial Dysfunctional Properties of Nano-Selenium in vitro and in vivo of Hyperhomocysteinemic Rats

doi: 10.2147/IJN.S255392

Figure Lengend Snippet: Hcy-mediated GPX1 and GPX4 inhibition and apoptosis were prevented by Nano-Se treatment. ( A ) Expressions of GPX 1 and GPX4 in HUVECs with Hcy incubation (1.5 mM) at given time points, as assessed by Western blot analysis. The representative Western blot results were shown, with β-actin expression as an internal control. The experiments were performed three times. ( B ) Determination of activities of GPX1 and GPX4 in HUVECs with Hcy incubation (1.5 mM) for 20 hours, detected by a coupled enzymatic assay. The histograms show the mean ± SEM of three separate experiments (*p<0.05). ( C ) Effect of GPX1 or GPX4 overexpression on HUVEC viability with Hcy for 24 hours. The cells were transfected with empty vector pCMV6 or pCMV-GPX1 or pCMV-GPX4 for 24 hours, followed by 1.5 mM Hcy treatment for another 24 hours. Cell viability was measured using annexin V/PI double staining. Representative dot plots of a CLL sample are shown, with numbers indicating the percentage of viable cells (annexin V/PI double negative). ( D ) Effect of Nano-Se on expressions of GPX1 and GPX4 in HUVECs with Hcy for 20 hours. The cells were preincubated with 100 nM or 500 nM Nano-Se for 8 hours before 1.5 mM Hcy was added. The expressions of GPX1 and GPX4 were assessed by Western blot analysis. The representative Western blot results were shown, with β-actin expression as an internal control. The experiments were performed three times. ( E ) Effect of Nano-Se on activities of GPX1 (left panel) and GPX4 (right panel) in HUVECs with Hcy for 20 hours. The cells were preincubated with 100 nM or 500 nM Nano-Se for 8 hours before 1.5 mM Hcy was added. The enzyme activities were examined by a coupled enzymatic assay. The histograms show the mean ± SEM of three separate experiments, each measured in triplicate (*p<0.05, **p<0.01). Abbreviations: Ctrl, control; Hcy, homocysteine; GPX1, glutathione peroxidase 1; GPX4, glutathione peroxidase 4; GPX1 OE, GPX1 overexpression; GPX4 OE, GPX4 overexpression.

Article Snippet: Briefly, 1×106 cells were transfected with 2 μg of GPX1 or GPX4 expression vector DNA (Origene, MD, USA) or empty vector (as control, Origene, Rockville, MD, USA) per cuvette in 100 μL of HUVEC Nucleofector solution using the A034 setting on the nucleofector.

Techniques: Inhibition, Incubation, Western Blot, Expressing, Enzymatic Assay, Over Expression, Transfection, Plasmid Preparation, Double Staining

Journal: International Journal of Nanomedicine

Article Title: Anti-Oxidant and Anti-Endothelial Dysfunctional Properties of Nano-Selenium in vitro and in vivo of Hyperhomocysteinemic Rats

doi: 10.2147/IJN.S255392

Figure Lengend Snippet: Effect of Nano-Se on vascular GPX expression, ROS and endothelial mitochondrial damage in hyperhomocysteinemic rats. ( A ) Expression of GPX 1 and GPX4 in the aorta of NR, HcyR and HcyR+Nano-Se as assessed by Western blot analysis. The representative Western blot results were shown. ( B ) Dihydroethidium (DHE) staining of the aorta of NR, HcyR and HcyR+Nano-Se. The arrows indicate endothelial cells. L, Lumen ( C ) TBARS content in the aorta of NR, HcyR and HcyR+Nano-Se. Representative histograms of three separate experiments are shown (*p<0.05; **p<0.01). ( D ) Ultrastructure of aortic endothelial cells of NR, HcyR and HcyR+Nano-Se. The representative transmission electron microscopic (TEM) images were shown. The arrows indicate mitochondria structures. Abbreviations: NR, normal rat; HcyR, hyperhomocysteinemic rat; HcyR+Nano-Se, hyperhomocysteinemic rat treated with Nano-Se; SHR, spontaneously hypertensive rat; SHHcyR, spontaneously hypertensive hyperhomocysteinemic rat; SHHcyR+Nano-Se, spontaneously hypertensive hyperhomocysteinemic rat treated with Nano-Se.

Article Snippet: Briefly, 1×106 cells were transfected with 2 μg of GPX1 or GPX4 expression vector DNA (Origene, MD, USA) or empty vector (as control, Origene, Rockville, MD, USA) per cuvette in 100 μL of HUVEC Nucleofector solution using the A034 setting on the nucleofector.

Techniques: Expressing, Western Blot, Staining, Transmission Assay

Journal: International Journal of Nanomedicine

Article Title: Anti-Oxidant and Anti-Endothelial Dysfunctional Properties of Nano-Selenium in vitro and in vivo of Hyperhomocysteinemic Rats

doi: 10.2147/IJN.S255392

Figure Lengend Snippet: Nano-Se, selenite and SeMet increased GPX1 and GPX4 activities and protected HUVECs against Hcy-induced oxidative stress and apoptosis. ( A ) Effects of selenite, SeMet and Nano-Se on the activities of GPX1 and GPX4 in HUVECs with Hcy. The cells were pre-treated with 100 nM or 500 nM of selenite, SeMet or Nano-Se for 8h before 1.5 mM Hcy was added, and the enzyme activities were examined by a coupled enzymatic assay. The histograms show the mean ± SEM of three separate experiments ( § p<0.05 vs Medium; *p<0.05, **p<0.01, ***p<0.001 vs Hcy treatment; # p<0.05 and ## p<0.01 vs Hcy+SeMet treatment). ( B ) Determination of cellular ROS in HUVECs with Hcy in the presence or absence of selenite, SeMet or Nano-Se for 16 hours, detected by flow cytometry analysis. The cells were pre-treated with 500 nM selenite, SeMet or Nano-Se 8 hours before 1.5 mM Hcy was added. Representative histograms of three separate experiments are shown. ( C ) Determination of mitochondrial transmembrane potential in HUVECs with Hcy in the presence or absence of selenite, SeMet or Nano-Se for 20 hours, detected by flow cytometry analysis. The cells were pre-treated with 500 nM selenite, SeMet or Nano-Se 8 hours before 1.5 mM Hcy was added. Representative histograms are shown. The numbers indicate the gating of the subpopulation of cells exhibiting loss of mitochondrial transmembrane potential. The experiments were performed three times. ( D ) Effect of selenite, SeMet or Nano-Se on HUVEC viability with Hcy for 24 hours. The cells were pre-treated with 500 nM selenite, SeMet or Nano-Se 8 hours before 1.5 mM Hcy was added. Cell viability was measured using annexin V/PI double staining. Representative dot plots of a CLL sample are shown, with numbers indicating the percentage of viable cells (annexin V/PI double negative). The experiments were performed three times. Abbreviations: Ctrl, control; Hcy, homocysteine; SeMet, selenomethionine.

Article Snippet: Briefly, 1×106 cells were transfected with 2 μg of GPX1 or GPX4 expression vector DNA (Origene, MD, USA) or empty vector (as control, Origene, Rockville, MD, USA) per cuvette in 100 μL of HUVEC Nucleofector solution using the A034 setting on the nucleofector.

Techniques: Enzymatic Assay, Flow Cytometry, Double Staining

Journal: The Journal of Cell Biology

Article Title: The α6β4 integrin promotes resistance to ferroptosis

doi: 10.1083/jcb.201701136

Figure Lengend Snippet: Matrix-deprived cells exhibit increased lipid peroxidation and an inability to sustain GPX4 expression in the absence of the α6β4-integrin. (A) GPX4 mRNA expression was quantified by qPCR in control and β4-depleted MCF-10A and SUM-159 cells under adherent or 2-h matrix-deprived conditions. (B) Expression of GPX4 was assessed by immunoblotting in vector control, β4-depleted, and β4-rescued cells after 2 h of detachment. Relative densitometry values are shown. (C) Lipid peroxidation was quantified using the MDA assay in control and β4-depleted, MCF-10A and SUM-159 cells under adherent or 4-h matrix-deprived conditions. (D) GPX enzyme activity was assayed in control and β4-depleted, MCF-10A and SUM-159 cells under adherent or 4-h matrix-deprived conditions. (E) Control and β4-depleted SUM-159 cells were transfected with either a vector control or a GPX4 expression vector. GPX4 mRNA expression was quantified by qPCR. (F) Control and β4-depleted MCF10-A and SUM-159 cells that had been transfected with either a vector control or a GPX4 expression vector were detached for 24 h, and the number of viable cells was quantified. (G) Control and β4-depleted cells that had been transfected with either a vector control or a GPX4 expression vector were detached for 24 h with in the presence of either DMSO or 10 µM erastin, and the number of viable cells was quantified. Experiments were performed independently three times and a representative experiment is shown. The bars in graphs represent means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Article Snippet: To express GPX4, a plasmid construct for GPX4 was purchased from OriGene (RC208065).

Techniques: Expressing, Control, Western Blot, Plasmid Preparation, Multiple Displacement Amplification, Activity Assay, 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: 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: Gpx4 -flox mice ( Gpx4 fl/fl , C57BL/6Smoc, NM-CKO-200097, purchased from Gempharmatech Co., Ltd., China) containing loxP sites flanking the second and fourth exons of Gpx4 were bred for two generations with granulosa cell-specific Cre driver mice, Cyp19a1 -Ires-Cre (Cyp19a1-Cre, C57BL/6Cya, C001382, Cyagen Biosciences Inc.).

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: Gpx4 -flox mice ( Gpx4 fl/fl , C57BL/6Smoc, NM-CKO-200097, purchased from Gempharmatech Co., Ltd., China) containing loxP sites flanking the second and fourth exons of Gpx4 were bred for two generations with granulosa cell-specific Cre driver mice, Cyp19a1 -Ires-Cre (Cyp19a1-Cre, C57BL/6Cya, C001382, Cyagen Biosciences Inc.).

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: Gpx4 -flox mice ( Gpx4 fl/fl , C57BL/6Smoc, NM-CKO-200097, purchased from Gempharmatech Co., Ltd., China) containing loxP sites flanking the second and fourth exons of Gpx4 were bred for two generations with granulosa cell-specific Cre driver mice, Cyp19a1 -Ires-Cre (Cyp19a1-Cre, C57BL/6Cya, C001382, Cyagen Biosciences Inc.).

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: Gpx4 -flox mice ( Gpx4 fl/fl , C57BL/6Smoc, NM-CKO-200097, purchased from Gempharmatech Co., Ltd., China) containing loxP sites flanking the second and fourth exons of Gpx4 were bred for two generations with granulosa cell-specific Cre driver mice, Cyp19a1 -Ires-Cre (Cyp19a1-Cre, C57BL/6Cya, C001382, Cyagen Biosciences Inc.).

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: Gpx4 -flox mice ( Gpx4 fl/fl , C57BL/6Smoc, NM-CKO-200097, purchased from Gempharmatech Co., Ltd., China) containing loxP sites flanking the second and fourth exons of Gpx4 were bred for two generations with granulosa cell-specific Cre driver mice, Cyp19a1 -Ires-Cre (Cyp19a1-Cre, C57BL/6Cya, C001382, Cyagen Biosciences Inc.).

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: Gpx4 -flox mice ( Gpx4 fl/fl , C57BL/6Smoc, NM-CKO-200097, purchased from Gempharmatech Co., Ltd., China) containing loxP sites flanking the second and fourth exons of Gpx4 were bred for two generations with granulosa cell-specific Cre driver mice, Cyp19a1 -Ires-Cre (Cyp19a1-Cre, C57BL/6Cya, C001382, Cyagen Biosciences Inc.).

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: Gpx4 -flox mice ( Gpx4 fl/fl , C57BL/6Smoc, NM-CKO-200097, purchased from Gempharmatech Co., Ltd., China) containing loxP sites flanking the second and fourth exons of Gpx4 were bred for two generations with granulosa cell-specific Cre driver mice, Cyp19a1 -Ires-Cre (Cyp19a1-Cre, C57BL/6Cya, C001382, Cyagen Biosciences Inc.).

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: Gpx4 -flox mice ( Gpx4 fl/fl , C57BL/6Smoc, NM-CKO-200097, purchased from Gempharmatech Co., Ltd., China) containing loxP sites flanking the second and fourth exons of Gpx4 were bred for two generations with granulosa cell-specific Cre driver mice, Cyp19a1 -Ires-Cre (Cyp19a1-Cre, C57BL/6Cya, C001382, Cyagen Biosciences Inc.).

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

Journal: Nature Aging

Article Title: Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis

doi: 10.1038/s43587-025-00902-5

Figure Lengend Snippet: Muscle stem cells were freshly isolated and plated for 4 h prior being treated with the ferroptosis-inducing compounds along with Ferrostatin (Fer1), a potent and selective inhibitor of ferroptosis. a-d , Erastin (A, B) and RSL3 (C, D) dose and time effects on Muscle stem cells. Cell viability (A, C) was quantified by DAPI integration and increased ROS was quantified by MitoSOX CTCF (B, D). e-g , Optimal treatment for Erastin (10uM) and RSL3 (1uM) for 24 h caused decreased viability (E; data reported as mean of individual experiment), increased ROS (F; data reported as individual cell intensity [CTCF] from 3 different experiments) and lipid peroxidation (G; data reported as mean of individual experiment). Fer1 rescued the effects of Erastin and RSL3, as it traps lipid radicals and lipid ROS that are involved in the induction of ferroptosis in Muscle stem cells. h, i ) Immunoblot and quantification for GPX4 relative to Actin following treatments. j ) Optimal dose for Kmt5a catalytic inhibitor UNC0379 was determined over 24 hours period, with 4uM effectively reducing H4K20me1 beyond detectable levels. k ) UNC0379 treatment (4uM) resulted in cell death initiating at 24 h with no detectable surviving cell past 72 h. Data are presented as mean ± standard deviation, n = 3 independent experiments. l ) Transcript levels of ferroptosis-associated genes Gpx4, Rgs4, Ptgs2, Hmox1 in response to RSL3. Gene expression was normalized to the average levels of B2M, TBP, and PPIA, and are reported as normalized fold-change ± s.d.

Article Snippet: For Gpx4 overexpression, we used Gpx4 ( BC106147 ) Mouse Tagged ORF Clone (cat. no. MG201418 ; OriGene).

Techniques: Isolation, Western Blot, Standard Deviation, Gene Expression

Journal: Nature Aging

Article Title: Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis

doi: 10.1038/s43587-025-00902-5

Figure Lengend Snippet: a , b , GPX4 immunostaining in TA muscles of WT ( a ) and Kmt5a KO ( b ) mice ( n = 5 mice per group, one experiment). Arrows, MuSCs; yellow arrow, MuSCs in iron-rich pocket (~50%). c , Quantification of MuSCs with high levels of labile iron (Fe 2+ ) (mean ± s.e.m., n = 5 mice per condition). d , Representative electron micrographs of a WT quiescent MuSC and a Kmt5a KO MuSC showing features of activation and ferroptosis. e , ICP-MS quantification of elemental iron in MuSCs. Total iron was normalized to cell numbers. Data points are reported as average of replicate ( n = 5 mice per condition). f , g , Quantification of lipid peroxidation in MuSCs. Flow cytometry plot shows a shift in 510-nm signals in mutant MuSCs. Inverted ratiometric signals of 590 nm/510 nm were calculated to report lipid peroxidation in each cell ( g ); data points are reported as average of replicate ( n = 4 mice per condition). h , qPCR for Gpx4 , Rgs4 and Ptgs2 . i , j , Immunoblot and quantification for KMT5a, H4K20me1 and GPX4 with DMSO or Kmt5a catalytic inhibitor (Kmt5a i ). Kmt5a was normalized to GAPDH; H4K20me1 was normalized to histone H3; and GPX4 was normalized to actin ( n = 3 mice per condition). Data are reported as normalized intensity ± s.d. k , qPCR quantification of ferroptosis markers Gpx4 , Rgs4 , Ptgs2 and Hmox1 in response to Kmt5a i . For qPCR data, gene expression was normalized to the average levels of B2M , TBP and PPIA and is reported as normalized fold change ± s.d. ( n = 3 mice per condition). l , m , Quantification of lipid peroxidation in response to drug treatments. Histogram shows the intensity of ratiometric signal (590 nm/510 nm) for the lipid peroxidation probe in live cells ( l ). Violin plots represent the inverted ratiometric signal normalized to vehicle ( m ). For all violin plots, data represent biological replicates. Statistical analyses were performed using two-sided Welch’s t -test ( e , j , k ) and one-way ANOVA ( g , h , l , m ), and exact P values and adjusted P values ( q ) are reported in the figure. wks, weeks; WT, wild-type; im, immediate.

Article Snippet: For Gpx4 overexpression, we used Gpx4 ( BC106147 ) Mouse Tagged ORF Clone (cat. no. MG201418 ; OriGene).

Techniques: Immunostaining, Muscles, Activation Assay, Flow Cytometry, Mutagenesis, Western Blot, Gene Expression

Journal: Nature Aging

Article Title: Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis

doi: 10.1038/s43587-025-00902-5

Figure Lengend Snippet: ( a, b ) Quantitative PCR (A) and immunoblot (B) analysis showing the levels of Gpx4 mRNA and protein in myoblasts treated with Kmt5a siRNA, with or without Kmt5a re-expression. B shows the whole WB membrane Data are presented as mean ± s.d. ( c, d ) Analysis of Kmt5a (C) and H4K20me1 (D) occupancy around the Gpx4 transcription start site (TSS) in control and Kmt5a-silenced myoblasts, with or without Kmt5a re-expression. Data are presented as % input ± s.d. from three independent experiments. ( e ) Lipid peroxidation analysis in control and Kmt5a-silenced myoblasts. Data are presented as mean ± s.e.m. from three independent experiments. ( f-i ) Quantitative PCR (F), immunoblot (G), cell viability (H), and lipid peroxidation (I) analysis in myoblasts treated with a Kmt5a inhibitor, with or without Gpx4 overexpression. Statistical analyses were performed using one-way ANOVA (A, E, F, H and I), and exact p-values are reported in the figure.

Article Snippet: For Gpx4 overexpression, we used Gpx4 ( BC106147 ) Mouse Tagged ORF Clone (cat. no. MG201418 ; OriGene).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Membrane, Control, Over Expression

Journal: Nature Aging

Article Title: Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis

doi: 10.1038/s43587-025-00902-5

Figure Lengend Snippet: a , b , H4K20me1 CUT&Tag in adult and aged MuSCs. a , Heatmap of RPKM-normalized sequencing reads centered on TSSs near H4K20me1 sites unique to young and aged MuSCs or shared between them. The numbers of TSSs in each group are labeled. b , Metaplot comparison of RPKM averaged in 50-bp bins around all TSSs. c , Transcriptional and H4K20me1 changes for ferroptosis genes. Heatmaps are organized by descending gene expression in aged MuSCs and are reported as fold change of z -score for RNA-seq and H4K20me1 CUT&Tag. RNA-seq and CUT&Tag were performed using different sets of mice and different times ( n = 3 mice per condition, per experiment). d , H4K20me1 signal at Gpx4 gene. FC, fold change; RPKM, reads per kilobase per million mapped reads.

Article Snippet: For Gpx4 overexpression, we used Gpx4 ( BC106147 ) Mouse Tagged ORF Clone (cat. no. MG201418 ; OriGene).

Techniques: Sequencing, Labeling, Comparison, Gene Expression, RNA Sequencing

Journal: Nature Aging

Article Title: Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis

doi: 10.1038/s43587-025-00902-5

Figure Lengend Snippet: a – c , scRNA workflow: adult (blue) and aged (orange) MuSCs were identified and plotted with UMAP ( a ). Unbiased clustering separated adult and aged cells into four clusters ( b ) prior to running pseudotime analysis to assess cell trajectory ( c ). d , Enrichment analysis of 500 most altered genes as a function of velocity and pseudotime trajectory highlighting ‘ferroptosis’ and ‘glutathione metabolism’ (red underlined). Statistical significance: Fisherʼs exact test followed by Benjamini–Hochberg correction. e , Dot plot for Louvain metadata clusters of MuSCs showing average expression and percent expression per cell for key genes. f , Donut plot representing a selection of MuSC fate based on flow cytometry analysis ( n = 5 mice per condition). Live cells were negative for all markers; senescent cells were SPiDER + ; apoptotic cells were Annexin V + ; ferroptotic cells were Lipid Peroxidation High ; other cells were DAPI + but negative for other markers. g , Lipid Peroxidation high MuSCs (Aged Fe ) were sorted for mRNA analysis to confirm a ferroptotic signature similar to cluster 3. h , Representative picture of an aged MuSC with intracellular labile iron and niche GPX4 ( n = 2 separate experiments). i , j , Abundance of total elemental iron per cell ( i ) and lipid peroxidation ( j ) in adult and aged MuSCs. To avoid additional introduction of cell stress bias and to validate lipid peroxidation changes in aged cells, we used a glutathione depletion mouse model (Nrf2 KO ;GCLC KO ) to naturally increase lipid peroxidation, therefore bypassing the need for a compound (that is, RSL3) or iron overload. k , Lipid peroxidation in MuSCs. Cells were isolated from adult and aged mice ( n = 5 male mice per condition), plated for 4 hours and harvested 20 hours after treatment. l – o , MuSC survival ( m ), myogenic potential ( o ) and fusion competence ( n ) in response to radical trapping drug Fer1. For all violin plots, data represent biological replicates. Statistical analyses were performed using two-sided Welch’s t -test ( g , i , j ), one-way ANOVA ( m – o ) and two-way ANOVA ( k ), and exact P values and adjusted P values are reported in the figure. Avg., average; exp., expression; NS, not significant; Veh, vehicle.

Article Snippet: For Gpx4 overexpression, we used Gpx4 ( BC106147 ) Mouse Tagged ORF Clone (cat. no. MG201418 ; OriGene).

Techniques: Expressing, Selection, Flow Cytometry, Isolation

Journal: Nature Aging

Article Title: Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis

doi: 10.1038/s43587-025-00902-5

Figure Lengend Snippet: a ) Schematic representation of the experimental design for aged plasma transfusion. Young mice were intravenously injected with aged plasma over a specified period to mimic chronic exposure. b ) Quantification of MuSC pool size following chronic aged plasma exposure. c-e ) Analysis of MuSCs sorted from aged plasma-treated mice. Flow cytometry analysis showing the loss of H4K20me1 (C) and repression of Kmt5a (E). (D) Lipid peroxidation analysis indicating increased lipid peroxidation in aged plasma-treated MuSCs. (E) qPCR analysis showing the repression of Gpx4 in MuSCs exposed to aged plasma. All effects were rescued by neutralizing antibodies against Ccr2-ligands. Data are presented as mean ± s.e.m. from three independent experiments with n = 10 male mice per conditions. Statistical analyses were performed using one-way ANOVA and p-values are reported in the figure.

Article Snippet: For Gpx4 overexpression, we used Gpx4 ( BC106147 ) Mouse Tagged ORF Clone (cat. no. MG201418 ; OriGene).

Techniques: Clinical Proteomics, Injection, Flow Cytometry

Journal: Nature Aging

Article Title: Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis

doi: 10.1038/s43587-025-00902-5

Figure Lengend Snippet: a ) Schematic representation of the experimental design. Young mice were chronically injected with plasma derived from aged mice treated with Saline or Bindarit for 12 months to assess potential rejuvenating effects. b ) Quantification of MuSC pool size in young mice following chronic plasma exposure. Plasma from Bindarit-treated aged mice maintained a normal MuSC pool comparable to control mice, unlike plasma from untreated aged mice. c ) Assessment of skeletal muscle regeneration in young mice treated with aged Bindarit plasma, showing normal regeneration similar to controls. d-g ) Analysis of MuSCs sorted from young mice exposed to aged Bindarit plasma. (D) Flow cytometry analysis showing levels of H4K20me1. (E) Lipid peroxidation analysis indicating normal lipid peroxidation levels. qPCR analysis showing normalized expression levels of Kmt5a (F) and Gpx4 (G). Data are presented as mean ± s.e.m. Statistical analyses were performed using one-way ANOVA (B-F), and p-values are indicated in the figure.

Article Snippet: For Gpx4 overexpression, we used Gpx4 ( BC106147 ) Mouse Tagged ORF Clone (cat. no. MG201418 ; OriGene).

Techniques: Injection, Clinical Proteomics, Derivative Assay, Saline, Control, Flow Cytometry, Expressing

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: FECD surgical tissues show key markers of ferroptosis. ( A ) Heatmap with hierarchical clustering for 211 genes from the FerrDB database that includes known driver, suppressor, and marker ferroptosis genes that were expressed in the RNA-Seq datasets. For each plot, “pearson” was used for the clustering distance and “complete” for the hierarchical clustering method. The location where the representative dataset was collected (Mayo, Russia, or UTSW) and mutation type (Control, no TCF4 repeats [No_Rep] or TCF repeats [TCF4_Rep]) are shown for each sample. ( B ) FSP1 mRNA and protein expression in FECD and control tissues. (C) FTH mRNA expression in control and FECD tissues. (D) GPX4 mRNA expression in FECD and control human tissues. (E) Ferroportin ( FPN1 ) mRNA and protein expression in FECD and control tissues. ( F ) FTL mRNA expression in control and FECD tissues. ( G ) TFR1 mRNA and protein expression in control and FECD surgical tissues. ( H ) Representative immunohistochemistry images of TFR1 localization in non-FECD and FECD donor cornea tissues. (I) 4-HNE protein expression in human surgical samples from patients with FECD (n = 8). All data of mRNA and protein expression are shown as mean ± SEM for n = 12 (Control tissues, 8 pools of 3, each pool contained 3 tissues) and n = 24 (FECD tissues, 8 pools of 3, each pool contained 3 tissues). All the statistical comparisons were conducted using two-tailed, unpaired Student's t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Relative gene expression is normalized by β-actin . (J) Cytosolic Fe 2+ in primary CECs isolated from healthy human donor corneas (n = 11, each cornea divided into 2 sections) and FECD surgical explants (n = 7). Data are shown as mean ± SEM; ∗∗p < 0.01, Student's t-test.

Article Snippet: 0.6 μg of total protein was loaded per capillary (DM-TP01, Protein Simple) and lysates were probed with antibodies directed at 4-HNE (STA-035, Cell Biolabs), GPX4 (MAB5457-SP, R&D Systems), NRF2 (PA5-14144, Invitrogen), FSP-1 (20886-1-AP, Proteintech), Ferroportin/SLC40A1 (PA5-G4232, Invitrogen) and TFR1 (MABS1982, Millipore) proteins.

Techniques: Marker, RNA Sequencing, Mutagenesis, Control, Expressing, Immunohistochemistry, Two Tailed Test, Gene Expression, Isolation

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: FECD primary and immortalized cell cultures show key marker s of ferroptosis. (A) TFR1 mRNA expression in non-FECD and FECD donor expanded TCF4 repeat expansion primary cells. ( B ) FSP1 mRNA expression in primary cells. ( C ) GPX4 mRNA expression in human expanded TCF4 repeat expansion primary cells. (D) FTH mRNA expression in non-FECD and FECD donor expanded TCF4 repeat expansion primary cells. (E) FTL mRNA expression in non-FECD and FECD donor primary cells. (F) Representation of median of the fluorescence of DHE showing significant difference in ROS between indicated cells. DHE (FL2 fluorescence) peak of F35T cells shifts to right when compared to B4G12 cells. (G) Representative confocal images showing fluorescence of DHE indicating ROS in the indicated cell lines. (H) Mitochondrial ROS quantified by MitoROS 580 dye in the indicated cells. Data are shown as mean ± SEM; n = 3; ∗∗∗∗p < 0.0001, one-way ANOVA, followed by Tukey's post-hoc test. AMA indicates antimycin-A . (I) GPX4 mRNA and protein expression in HCEC-B4G12 and F35T cells. ( J ) Basal level of lipid peroxidation in HCEC-B4G12 and F35T cells quantified by C11-BODIPY fluorescent probe using flow cytometry. Comparisons of median fluorescence of C11-BODIPY detected in HCEC-B4G12 and F35T cells (10,000 cells). Data are shown as mean ± SEM; n = 3; ∗∗∗∗p < 0.0001, Student's t-test. C11-BODIPY (FL1 fluorescence) peak of F35T cells shifts to right when compares to B4G12 cells. ( K ) Representative confocal images showing fluorescence of reduced and oxidized dye in the indicated cell lines. ( L ) 4-HNE protein expression in HCEC-B4G12 and F35T cells. All data of mRNA and protein expression are shown as mean ± SEM for n = 5–9 (B4G12), n = 5–7 (F35T) and n = 4 (both non-FECD and FECD donor primary cells). All the statistical comparisons were conducted using two-tailed, unpaired Student's t-test, ∗∗∗p < 0.001. Relative gene expression is normalized by β -actin.

Article Snippet: 0.6 μg of total protein was loaded per capillary (DM-TP01, Protein Simple) and lysates were probed with antibodies directed at 4-HNE (STA-035, Cell Biolabs), GPX4 (MAB5457-SP, R&D Systems), NRF2 (PA5-14144, Invitrogen), FSP-1 (20886-1-AP, Proteintech), Ferroportin/SLC40A1 (PA5-G4232, Invitrogen) and TFR1 (MABS1982, Millipore) proteins.

Techniques: Marker, Expressing, Fluorescence, Flow Cytometry, Two Tailed Test, Gene Expression

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: Summary of the molecular mechanism of ferroptosis in FECD. Iron enters the cell in ferric form via TFR1-mediated endocytosis. Ferritin stores the excess iron in ferric form, which is nontoxic. The ferric form of iron gets converted to the ferrous form in endosomes. When labile, ferrous iron gets released into the cytosol, it causes lipid peroxidation via Fenton chemistry. UVA irradiation can cause iron release from ferritin which increases the labile iron pool in the cytosol, as well as increases iron-mediated lipid peroxidation, a process known as ferroptosis. GPX4 is the key regulator of ferroptosis, preventing occurrence through scavenging lipid peroxides and reactive oxygen species (ROS). In this study, RSL3 was used to block GPX4 to induce ferroptosis. Ubiquinol, the reduced and active form of coenzyme Q10, is a potent ferroptosis inhibitor that works by scavenging ROS and modulating iron metabolism. Ubiquinol is an essential participant in the FSP1-CoQ10-NAD(P)H pathway, an independent system working in parallel with GPX4 and glutathione to suppress lipid peroxidation and ferroptosis by supporting FSP1 function. Other molecules like DFO and artesunate can prevent ferroptosis by quenching labile toxic ferrous iron, however, are not solely as effective as ubiquinol in preventing ferroptosis.

Article Snippet: 0.6 μg of total protein was loaded per capillary (DM-TP01, Protein Simple) and lysates were probed with antibodies directed at 4-HNE (STA-035, Cell Biolabs), GPX4 (MAB5457-SP, R&D Systems), NRF2 (PA5-14144, Invitrogen), FSP-1 (20886-1-AP, Proteintech), Ferroportin/SLC40A1 (PA5-G4232, Invitrogen) and TFR1 (MABS1982, Millipore) proteins.

Techniques: Irradiation, Blocking Assay