Journal: Cell communication and signaling : CCS

Article Title: Inhibition of tubular epithelial cells ferroptosis alleviates renal interstitial fibrosis by reducing lipid hydroperoxides and TGF-β/Smad signaling.

doi: 10.1186/s12964-025-02068-4

Figure Lengend Snippet: Fig. 6 TGF-β/Smad signaling activation during UUO is inhibited by ferroptosis deficiency in GPX4-overexpressing mice. a Western blot analysis of the expression of TGF-β, p-Smad2, and p-Smad3. b Relative transcript levels of genes related to profibrotic cytokines (TGF-β, CTGF, PDGFB, and FGF2) in UUO mice overexpressing GPX4. For all panels, the data are presented as the means ± SDs. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001, ns indicates no significance. Statistical analysis was performed via two-way analysis of variance (ANOVA) with the Bonferroni post hoc correction

Article Snippet: Additional gene transfection studies included the use of a GPX4 plasmid (origene, catalog RC208065; 5 μg) in cells transfected with Lipofectamine 3000 (Thermo Fisher Scientific, MA).

Techniques: Activation Assay, Western Blot, Expressing

Journal: Cells

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

doi: 10.3390/cells12101428

Figure Lengend Snippet: Figure 2. 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

Journal: Cells

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

doi: 10.3390/cells12101428

Figure Lengend Snippet: Figure 3. 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: bioRxiv

Article Title: Sodium Aurothiomalate Induces Ferroptosis by Targeting GPX4 via Gold-Dependent Thiomalate Covalent Modification

doi: 10.1101/2025.11.13.687868

Figure Lengend Snippet: Modulation of ATM-induced ferroptosis by ferroptosis inhibitors. a, SK-N-DZ cells treated with 100 μM ATM in the presence or absence of ferroptosis inhibitors (5 μM Fer-1, 1 μM DFO, 1 μM CPX, or 20 μM Zileuton). b-c, Effect of sodium selenite supplementation on cell viability following treatment with ATM or auranofin. d-e, Survival of SK-N-DZ cells treated with ATM or auranofin with or without overexpression of GPX4 or AIFM2 (FSP1). f, Western blots showing successful overexpression of AIFM2 and GPX4. Data represents SEM; *p<0.05, **p<0.01.

Article Snippet: To induce overexpression of GPX4 and AIFM2, individual gRNAs were cloned into the pXPR_502 vector (Addgene #96923) through restriction digestion of the respective lentivector with BsmBI (NEB, Cat. No. R0739).

Techniques: Over Expression, Western Blot

Journal: bioRxiv

Article Title: Sodium Aurothiomalate Induces Ferroptosis by Targeting GPX4 via Gold-Dependent Thiomalate Covalent Modification

doi: 10.1101/2025.11.13.687868

Figure Lengend Snippet: Gene and protein expression signature of ferroptosis upon ATM treatment: a, pathway analysis of SK-N-DZ cells treated with ATM for 24 hours. b, Transcriptional changes of ferroptosis markers of SK-N-DZ cells after 24 hours of treatment with ATM. ***P < 0.001; **P < 0.01. *P < 0.05. c-d, Protein level changes of ferroptosis markers in SK-N-DZ cells after 24 (c) or 48 hours (d) of treatment with media (left) or 200 μM ATM (right). e, Schematic representation of the proteins involved in selenium metabolism and ferroptotic pathways, whose expression is upregulated (green arrow) or downregulated (red arrow) upon 48 hours of treatment with ATM. Protein levels comparing control and ATM treatment after 48 hours are plotted as bars. ATM promotes degradation of GPX4, while other ferroptosis related proteins like LRP8, SLC7A11 and GR are increased. FSP1/AIFM2 was not detected in proteomics data, but immunoblotting (Supplementary Fig. 3d) also shows an upregulation. ****P < 0.0001 ***P < 0.001; **P < 0.01. *P < 0.05.

Article Snippet: To induce overexpression of GPX4 and AIFM2, individual gRNAs were cloned into the pXPR_502 vector (Addgene #96923) through restriction digestion of the respective lentivector with BsmBI (NEB, Cat. No. R0739).

Techniques: Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: Sodium Aurothiomalate Induces Ferroptosis by Targeting GPX4 via Gold-Dependent Thiomalate Covalent Modification

doi: 10.1101/2025.11.13.687868

Figure Lengend Snippet: ATM targets the GPX/GR system: a, Schematic representation of the enzyme-coupled assay for the measurement of GPX activity. b-c, The activity of purified recombinant Sec-containing human GPX4 was measured using an enzyme coupled assay with glutathione reductase (GR), reduced glutathione (GSH) and NADPH as shown in (a), in the absence and presence of a wide range of ATM (b) or Thiomalate (c) concentrations (0.2-1000 µM). The reaction was initiated by the addition of 0.5 mM (final concentration in well) Cumene hydroperoxide to the reaction mixture and the NADPH fluorescence (λ ex = 340 nm; λ em = 450 nm) was monitored over time. d, Four-parameter dose-response curve fit to n = 2 technical replicates and IC 50 calculations of ATM and Thiomalate. e-f, thermal shift assay with purified GPX4 at a final concentration of 1 μM and SYPRO orange dye, which binds to the hydrophobic patch of the protein once it is melted, resulting in a stronger fluorescence signal. g, Docking model for ATM in the region involving U46.

Article Snippet: To induce overexpression of GPX4 and AIFM2, individual gRNAs were cloned into the pXPR_502 vector (Addgene #96923) through restriction digestion of the respective lentivector with BsmBI (NEB, Cat. No. R0739).

Techniques: Activity Assay, Purification, Recombinant, Concentration Assay, Fluorescence, Thermal Shift Assay

Journal: bioRxiv

Article Title: Sodium Aurothiomalate Induces Ferroptosis by Targeting GPX4 via Gold-Dependent Thiomalate Covalent Modification

doi: 10.1101/2025.11.13.687868

Figure Lengend Snippet: Covalent modification of GPX4 by ATM leads to loss of enzymatic function and destabilization. a-c, Peptide fragmentation spectra of identified covalent modification sites of free Sec (U, green) and Cys (C, blue) residues. d-f, Quantification of modified peptides shown in a-c, respectively, upon incubation of the protein with equimolar or 5x excess of ATM. g-i, Proposed best docking poses for ATM in three different sites: U46, C66 and C148. Binding energy values as derived from the docking analysis are shown as insets. j-k, Proposed reaction mechanisms between ATM and the (Se)Cys sites of GPX4. Only covalent bonds with the thiomalate moiety could be detected. l, Bio-Layer Interferometry following association of nanodiscs loaded with negatively charged 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) to GPX4, without and in the presence of 1 nM, 200 nM, 1 µM and 100 µM ATM (black, orange, blue, cyan and green lines, respectively). The observed rates of GPX4-POPG interaction (kobs) indicate no effects at the presence of ATM up to 1 nM and considerably slower binding at ATM concentrations of 1 µM and similarly at 100 µM.. m-o, Thiomalate covalently bound to Sec46 modifies GPX4 stability. m, Root mean square fluctuations (RMSF) difference between GPX4 in the free (n) and bound to TML (o) proposed states. While some regions experience minor differences in atomic fluctuations, a greater change is observed for the region between residues 152 and 158 (light blue), corresponding to a disordered region connecting the beta-strand comprising residues 148 to 154 and the alpha-helix comprising residues 159 and 171.

Article Snippet: To induce overexpression of GPX4 and AIFM2, individual gRNAs were cloned into the pXPR_502 vector (Addgene #96923) through restriction digestion of the respective lentivector with BsmBI (NEB, Cat. No. R0739).

Techniques: Modification, Incubation, Binding Assay, Derivative Assay

Journal: Stem cell research & therapy

Article Title: MSCs promote the efferocytosis of large peritoneal macrophages to eliminate ferroptotic monocytes/macrophages in the injured endometria.

doi: 10.1186/s13287-024-03742-z

Figure Lengend Snippet: Fig. 1 Accumulation of large peritoneal macrophages following injury in the uteri. (A) Schematic of in vivo IUA mice model setup (n = 6–8). (B) The mRNA expression levels of TNF-α, IL-1β, and IL-6 in the endometria of mice were determined by qRT-PCR (normalised to β-actin). (C) Serum concentrations of TNF-α, IL-1β, and IL-6 were measured by ELISA. (D) Flow cytometry analysis for LPMs isolated from peritoneal cavity harvested at different time points after injury. Cells were pregated on CD11b+. (E) Flow cytometry analysis for LPMs isolated from uteri harvested at different time points after injury. (F) Im munohistochemical staining of MDA in the endometria of mice (scale bar: 100 μm). (G) Flow cytometry analysis for GPX4 expression of CD11b+F4/80med subsets in the uteri harvested at different time points after injury. (H) Schematic of in vivo LPMs depletion mice model setup (n = 6). (I) The mRNA expres sion levels of TNF-α, IL-1β, and IL-6 in the endometria of mice were determined by qRT-PCR (normalised to β-actin). (J) Serum concentrations of TNF-α, IL-1β, and IL-6 were measured by ELISA. (K) Flow cytometry analysis for LPMs isolated from peritoneal cavity harvested after 2-days injury. Cells were pre gated on CD11b+. (L) Flow cytometry analysis for LPMs isolated from uteri harvested after 2-days injury. (M) Flow cytometry analysis for GPX4 expression of CD11b+F4/80med subsets in the uteri harvested after 2-days injury. Values are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns denotes p > 0.05 (by unpaired Student’s t test)

Article Snippet: CD11b (Catalog # 101,206; Biolegend, San Diego, CA, USA), F4/80 (Catalog # 17-4801-82; Invitrogen, Carlsbad, CA, USA), CD102 (Catalog # 742,107; BD Pharmingen, San Jose, CA, USA), MHCII (Catalog # 12-5321-82; eBioscience, San Diego, CA, USA), GPX4 (Catalog # NBP3-08253 V; Novus Littleton, CO, USA) were used for labeling cells.

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Isolation, Staining

Journal: Stem cell research & therapy

Article Title: MSCs promote the efferocytosis of large peritoneal macrophages to eliminate ferroptotic monocytes/macrophages in the injured endometria.

doi: 10.1186/s13287-024-03742-z

Figure Lengend Snippet: Fig. 5 MSCs promote the efferocytosis and migration of LPMs to the injured uteri to repair the injured endometria. (A) Schematic of in vitro coculture system setup. (B, C) Efferocytosis of LPMs from each group was determined by (B) flow cytometry and (C) confocal microscope (scale bar: 50 μm). (D) Crystal violet staining of LPMs cocultured with MSCs or LPS (scale bar: 100 μm). (E) Schematic of in vivo experimental model design (n = 5–7). (F) Flow cytometry analysis for LPMs isolated from peritoneal cavity harvested after 7-days injury. Cells were pregated on CD11b+. (G) Flow cytometry analysis for LPMs isolated from uteri harvested after 7-days injury. (H) Flow cytometry analysis for GPX4 expression of CD11b+F4/80med subsets in the uteri harvested after 7-days injury. (I) HE, Masson’s trichrome staining, MDA, ER, and PR stainings of endometrial tissues obtained from mice of three groups. Scale bar indicates 100 μm. (J) The mRNA expression levels of TNF-α, IL-1β, and IL-6 in the endometria of mice were determined by qRT-PCR (normalised to β-actin). (K) Serum concentrations of TNF-α, IL-1β, and IL-6 were measured by ELISA. Values are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns denotes p > 0.05 (by unpaired Student’s t test)

Article Snippet: CD11b (Catalog # 101,206; Biolegend, San Diego, CA, USA), F4/80 (Catalog # 17-4801-82; Invitrogen, Carlsbad, CA, USA), CD102 (Catalog # 742,107; BD Pharmingen, San Jose, CA, USA), MHCII (Catalog # 12-5321-82; eBioscience, San Diego, CA, USA), GPX4 (Catalog # NBP3-08253 V; Novus Littleton, CO, USA) were used for labeling cells.

Techniques: Migration, In Vitro, Flow Cytometry, Microscopy, Staining, In Vivo, Isolation, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Stem cell research & therapy

Article Title: MSCs promote the efferocytosis of large peritoneal macrophages to eliminate ferroptotic monocytes/macrophages in the injured endometria.

doi: 10.1186/s13287-024-03742-z

Figure Lengend Snippet: Fig. 6 MSCs exert the effect of repairing the injured endometria through LPMs. (A) Schematic of in vivo experimental model design (n = 6–8). (B) Flow cytometry analysis for LPMs isolated from peritoneal cavity harvested after 7-days injury. Cells were pregated on CD11b+. (C) Flow cytometry analysis for LPMs isolated from uteri harvested after 7-days injury. (D) Flow cytometry analysis for GPX4 expression of CD11b+F4/80med subsets in the uteri harvested after 7-days injury. (E) HE staining, Masson’s trichrome staining, and MDA staining of endometrial tissues obtained from mice of four groups. Scale bar indicates 100 μm. (F) The mRNA expression levels of TNF-α, IL-1β, and IL-6 in the endometria of mice were determined by qRT-PCR (normalised to β-actin). (G) Serum concentrations of TNF-α, IL-1β, and IL-6 were measured by ELISA. Values are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (by unpaired Student’s t test)

Article Snippet: CD11b (Catalog # 101,206; Biolegend, San Diego, CA, USA), F4/80 (Catalog # 17-4801-82; Invitrogen, Carlsbad, CA, USA), CD102 (Catalog # 742,107; BD Pharmingen, San Jose, CA, USA), MHCII (Catalog # 12-5321-82; eBioscience, San Diego, CA, USA), GPX4 (Catalog # NBP3-08253 V; Novus Littleton, CO, USA) were used for labeling cells.

Techniques: In Vivo, Flow Cytometry, Isolation, Expressing, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Stem cell research & therapy

Article Title: MSCs promote the efferocytosis of large peritoneal macrophages to eliminate ferroptotic monocytes/macrophages in the injured endometria.

doi: 10.1186/s13287-024-03742-z

Figure Lengend Snippet: Fig. 7 MSCs promote the efferocytosis of LPMs by secreting STC-1 in vitro and in vivo. (A, B) The mRNA (A) and protein (B) levels of STC-1 in MSCs co cultured with LPMs stimulated with LPS for 24 h were determined by qRT-PCR and ELISA (normalised to GAPDH). (C, D) LPMs were incubated with non treated or Erastin-treated THP-1 cells in the presence of rhSTC-1 protein (100 ng/mL) for 4 h. Efferocytosis of LPMs from each group was determined by (C) flow cytometry and (D) confocal microscope (scale bar: 40 μm). (E, F) qRT-PCR analysis (E) and ELISA (F) showed the mRNA and protein levels of STC-1 in MSCs transfected with three interfering fragments of STC-1 (si-STC-1) for 24 h (normalised to GAPDH). (G, H) MSCs were transfected with siSTC-1 for 24 h before cocultured with LPMs, then LPMs were incubated with Erastin-treated THP-1 cells stained by CFSE for 4 h. Efferocytosis of LPMs from each group was determined by (G) flow cytometry and (H) confocal microscope (scale bar: 40 μm). (I) Schematic of in vivo experimental model design (n = 5–7). (J) Flow cytometry analysis for LPMs isolated from peritoneal cavity harvested after 7-days injury. Cells were pregated on CD11b+. (K) Flow cytometry analysis for LPMs isolated from uteri harvested after 7-days injury. (L) Flow cytometry analysis for GPX4 expression of CD11b+F4/80med subsets in the uteri harvested after 7-days injury. (M) HE staining, Masson’s trichrome staining, and MDA staining of endometrial tissues obtained from mice of four groups. Scale bar indicates 100 μm. (N) The mRNA expression levels of TNF-α, IL-1β, and IL-6 in the endometria of mice were determined by qRT-PCR (normalised to β-actin). (O) Serum concentrations of TNF-α, IL-1β, and IL-6 were measured by ELISA. Values are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns denotes p > 0.05 (by unpaired Student’s t test)

Article Snippet: CD11b (Catalog # 101,206; Biolegend, San Diego, CA, USA), F4/80 (Catalog # 17-4801-82; Invitrogen, Carlsbad, CA, USA), CD102 (Catalog # 742,107; BD Pharmingen, San Jose, CA, USA), MHCII (Catalog # 12-5321-82; eBioscience, San Diego, CA, USA), GPX4 (Catalog # NBP3-08253 V; Novus Littleton, CO, USA) were used for labeling cells.

Techniques: In Vitro, In Vivo, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Microscopy, Transfection, Staining, Isolation, Expressing

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: iScience

Article Title: TXNDC12 inhibits lipid peroxidation and ferroptosis

doi: 10.1016/j.isci.2023.108393

Figure Lengend Snippet: TXNDC12 inhibits ferroptosis independent on GPX4 (A) The protein expression of GPX4 was analyzed by western blot in indicated K562 cells with or without GPX4 overexpression. (B) Cell death analysis was performed on the indicated K562 cells with or without GPX4 overexpression in response to erastin (10 μM) or RSL3 (0.5 μM) for 4–24 h. (C) The levels of MDA were analyzed in the indicated K562 cells with or without GPX4 overexpression in response to erastin (10 μM) or RSL3 (0.5 μM) for 24 h. (D) The protein expression of TXNDC12 was analyzed by western blot in indicated WT and Gpx4 -knockout Pfa-1 cells with or without Txndc12 overexpression. (E) Cell death analysis was performed on the indicated Pfa-1 cells with or without Txndc12 overexpression. (F) The levels of MDA were analyzed in the indicated Pfa-1 cells at 72 h. (G) Cell death analysis was performed on the indicated Pfa-1 cells in response to erastin (5 μM) for 24 h. The data represent the means ± SD from three independent samples. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. ∗p < 0.05 versus WT group. (H) IP analysis was performed on K562 cells in response to erastin (10 μM) for 12 h. (I) Fluorescent calcein dye was entrapped within liposomes, which were subsequently exposed to Fe 2+ (10 μM) under conditions either with or without the presence of ferrostain-1 (1 μM), rGPX4 (200 nM), or rTXNDC1 (200 nM) for a duration of 20 min. The release of calcein from the liposomes was assessed by measuring changes in calcein fluorescence intensity. The data represent the means ± SD from three independent samples. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. ∗p < 0.05 versus Fe group.

Article Snippet: rGPX4 , OriGene , TP762179.

Techniques: Expressing, Western Blot, Over Expression, Knock-Out, Liposomes, Fluorescence

Journal: iScience

Article Title: TXNDC12 inhibits lipid peroxidation and ferroptosis

doi: 10.1016/j.isci.2023.108393

Figure Lengend Snippet:

Article Snippet: rGPX4 , OriGene , TP762179.

Techniques: Recombinant, Lysis, Bicinchoninic Acid Protein Assay, CCK-8 Assay, Transfection, Reporter Assay, Iron Assay, Multiple Displacement Amplification, Magnetic Beads, Mutagenesis, Chromatin Immunoprecipitation, Purification, Enzyme-linked Immunosorbent Assay, shRNA, Software