Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: D-EVs Counteract NPC Senescence by Suppressing Ferroptosis. (A) KEGG pathway analysis of DEGs in senescent NPCs following treatment with D-EVs or not. (B-C) GSEA plots showing significant enrichment of ferroptosis and cell cycle in senescent NPCs. (D-E) Heatmap quantification of key genes involved in ferroptosis and cell cycle. (F) Western blot analysis of key ferroptosis (GPX4, SLC7A11, ACSL4) and senescence (p21, P16) markers in NPCs following treatment with different experimental conditions. (G) Representative images of C11-BODIPY 581/591 staining to detect lipid peroxidation (green) in the control, TBHP, Era, Era + Fer-1, or TBHP + Fer-1 groups. (H-I) Quantitative assessment of malondialdehyde (MDA) levels (H) and glutathione (GSH) levels (I) in the control, TBHP, N-EVs, D-EVs, or D-EVs + Era groups. (J) Western blot analysis of key ferroptosis (GPX4, SLC7A11, ACSL4) and senescence (p21, P16) markers in NPCs following treatment with PBS, N-EVs, D-EVs, or D-EVs + Era. (K) Confocal analysis of GPX4 with IF staining in the control, TBHP, N-Evs, D-EVs, and D-EVs + Era group. (L) Flow cytometry analysis of cell cycle distribution in the above experimental conditions. Statistical comparisons were performed between the experimental group and the TBHP-induced group. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Western Blot, Staining, Control, Flow Cytometry

Journal: iScience

Article Title: LRP1 activated by AT2 cell-secreted MDK inhibits fibrotic ferroptosis in idiopathic pulmonary fibrosis

doi: 10.1016/j.isci.2026.116212

Figure Lengend Snippet: OTUB1 targets SLC7A11 and stabilizes its expression level (A) Molecular docking of OTUB1 and SCL7A11. (B and C) IP and co-IP assays of OTUB1 and SCL7A11 in human primary lung fibroblasts and 293T cells. ( n = 3 biological replicates for each group). (D) Laser confocal imaging of SLC7A11 and PTUB1 subcellular localization. ( n = 3 biological replicates for each group; Scale bars = 50 μm). (E) Protein structures of OTUB1 and SLC7A11. (F) Flag-tagged OTUB1-FL (1–272), OTUB1-M1 (1–85), OTUB1-M2 (86–195), or OTUB1-M3 (196–272) were transfected into HEK293T cells with Myc-SLCA11 and were then subjected to IP with anti-Flag (Left). Myc-tagged FL-SLC7A11 (1–501), SLC7A11-M1 (1–159), SLC7A11-M2 (160–291), or SLC7A11-M3 (292–501) were transfected into HEK293T cells with Flag-tagged FL-OTUB1 and were then subjected to IP with anti-Myc (Right). (G) Fibroblasts transfected with two independent OTUB1 shRNAs were treated with or without the proteasome inhibitor MG132 (20 μM, 8 h); subsequently, OTUB1 and SLC7A11 were assessed by western blotting. ( n = 3 biological replicates for each group). (H) Increasing concentrations of OTUB1 (WT) or OTUB1 (CA) were transfected into HEK293T cells, and SLC7A11 expression was assessed by western blotting. ( n = 3 biological replicates for each group). (I) Fibroblasts transfected with the shCtrl or shOTUB1-2 were treated with CHX (10 μg/mL) and collected at the indicated time points for western blotting. ( n = 3 biological replicates for each group). (J) Fibroblasts transfected with the vector control, OTUB1 (WT) or OTUB1 (CA) were treated with CHX (10 μg/mL) and collected at the indicated time points for western blotting. ( n = 3 biological replicates for each group).

Article Snippet: The structures of SLC7A11 (Protein DataBank code 7CCS) and OTBU1 (Protein DataBank code 4DDL) were used for docking simulations.

Techniques: Expressing, Co-Immunoprecipitation Assay, Imaging, Transfection, Western Blot, Plasmid Preparation, Control

Journal: iScience

Article Title: LRP1 activated by AT2 cell-secreted MDK inhibits fibrotic ferroptosis in idiopathic pulmonary fibrosis

doi: 10.1016/j.isci.2026.116212

Figure Lengend Snippet: OTUB1 deubiquitinates SLC7A11 and directly cleaves the Lys 48-polyubiquitin chain (A) Fibroblasts treated with 20 μM MG132 for 8 h were co-transfected with HA-Ub and shCtrl, shOTUB1-1, or shOTUB1-2, and cell lysates were subjected to IP with SLC7A11 antibody followed by IB with the indicated antibodies. ( n = 3 biological replicates for each group). (B) Fibroblasts treated with 20 μM MG132 for 8 h were co-transfected with Myc-SLC7A11, HA-Ub, and Flag-OTUB1 (WT) or Flag-OTUB1 (CA), and cell lysates were subjected to IP with Myc antibody followed by IB with the indicated antibodies. ( n = 3 biological replicates for each group). (C) Unubiquitylated or ubiquitylated His-SLC7A11 was incubated with GST-OTUB1 (WT) or GST-OTUB1 (CA) and HA-Ub coupled to glutathione Sepharose beads. His-SLC7A11 was subjected to IP with His beads followed by IB with antibodies against HA and His. ( n = 3 biological replicates for each group). (D) HEK293T cells were co-transfected with Myc-SLC7A11, Flag-OTUB1 (CA), and HA-Ub (Lys0), HA-Ub (Lys48), or HA-Ub (Lys63) plasmids, and the SLC7A11 poly-ubiquitylation linkage was analyzed. ( n = 3 biological replicates for each group). (E) Fibroblasts were co-transfected with Myc-SLC7A11, Flag-OTUB1, and HA-Ub (WT) or HA-Ub (Lys48R) plasmids, and the SLC7A11 poly-ubiquitylation linkage was analyzed. ( n = 3 biological replicates for each group). (F) Unubiquitylated or ubiquitylated His-SLC7A11 was incubated with GST-OTUB1 (WT) or GST-OTUB1 (CA) and HA-Ub coupled to glutathione Sepharose beads. His-SLC7A11 was subjected to IP with His beads followed by IB with antibodies against HA and His. ( n = 3 biological replicates for each group). (G) Fibroblasts transfected with WT or Lys48-Res were cultured for 72 h in the presence of control or sh-OTUB1-2. Cell lysates were analyzed by IB using antibodies against SLC7A11 and OTUB1. shOTUB1: small hairpin of OTUB1, shCtrl: small hairpin control. ( n = 3 biological replicates for each group).

Article Snippet: The structures of SLC7A11 (Protein DataBank code 7CCS) and OTBU1 (Protein DataBank code 4DDL) were used for docking simulations.

Techniques: Transfection, Incubation, Cell Culture, Control

Journal: iScience

Article Title: LRP1 activated by AT2 cell-secreted MDK inhibits fibrotic ferroptosis in idiopathic pulmonary fibrosis

doi: 10.1016/j.isci.2026.116212

Figure Lengend Snippet: SLC7A11 knockdown in fibroblasts partially reverses pulmonary fibrosis in vivo (A–C) Representative images of H&E staining (A), Masson staining (B), and IHC of hydroxyproline (C) for pulmonary tissues from BLM mice and BLM mice with OTUB1 overexpression and/or SLC7A11 knockdown ( n = 6 biological replicates for each group; mean ± SD; one way ANOVA; Scale bars = 200 μm). (D) Quantification of the concentrations of hydroxyproline from C and the Ashcroft score evaluating pulmonary fibrosis of mouse lungs ( n = 6 biological replicates for each group; mean ± SD; one way ANOVA; ∗ p < 0.05). (E–H) The protein levels of hub ferroptosis regulators (E), the level of lipid oxidation (F and G), the GSH/GSSG ratio and MDA level (H) in fibroblasts extracted from the mice. ( n = 6 biological replicates for each group; mean ± SD; one way ANOVA; Scale bars = 50 μm ∗ p < 0.05).

Article Snippet: The structures of SLC7A11 (Protein DataBank code 7CCS) and OTBU1 (Protein DataBank code 4DDL) were used for docking simulations.

Techniques: Knockdown, In Vivo, Staining, Over Expression

Journal: iScience

Article Title: Inflammatory CD4 + T cells can waive NRF2-dependent SLC7A11-mediated cystine uptake by using ASCT1

doi: 10.1016/j.isci.2026.115680

Figure Lengend Snippet: Regulation of lipid peroxidation is extrinsically regulated by the SLC7A11 pathway in CD4 + T cells (A) Statistical analysis of SLC7A11 protein expression assessed by flow cytometry (shown as mean fluorescent intensity (MFI). CD4 + T cells were either left unstimulated (control) or measured at a distinct time point after stimulation with anti-CD3/CD28 antibodies. N = 3, 3 independently performed experiments, a one-way ANOVA multiple comparison test was performed. The calculated power of this experiment is 0.84. (B) ΔCT of SLC7A11 mRNA expression in human CD4 + T cells with and without anti-CD3/CD28 stimulation. N = 13, 4 independently performed experiments. (C) Gene expression in CD4 + T cells from WT and VAV cre Keap fl/fl (Keap1-KO) mice was assessed by microarray analysis. Colors indicate significant upregulation (red), or downregulation (green) compared to WT. (D) ΔCT of SLC7A11 mRNA expression in CD4 + T cells of WT and Keap1-KO mice, either left unstimulated or stimulated with anti-CD3/CD28 antibodies, 4 independently performed experiments. The calculated power of this experiment is 0.99 for the stimulated and unstimulated groups. (E) N-fold SLC7A11 expression in human CD4 T cells, either transfected with a control siRNA (Ctrl) or NRF2 siRNA. 3 independently performed experiments were performed. (F) N-fold SLC7A11 mRNA expression in human CD4 T cells treated with 4-OI or vehicle (Ctrl) N = 6, 4 independently performed experiments. A paired Student's t-test was performed comparing the groups of B–F. Data are presented as mean, error bars present ± SEM for all the presented graphs in this figure. Values were considered significant if ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. N represents the number of biological replicates.

Article Snippet: SLC7A11 , F: 5′ TGA CTG GAGTCC CTG CGT AT 3′; R: 5′ TGT TCT GGT TAT TTT CTC CGA CA 3′ , eurofins.

Techniques: Expressing, Flow Cytometry, Control, Comparison, Gene Expression, Microarray, Transfection

Journal: iScience

Article Title: Inflammatory CD4 + T cells can waive NRF2-dependent SLC7A11-mediated cystine uptake by using ASCT1

doi: 10.1016/j.isci.2026.115680

Figure Lengend Snippet: Inhibition of SLC7A11 by SAS and erastin reduces the proliferation of T cells and the release of inflammatory cytokines (A) CD4 + T cells were labeled with cell proliferation dye and left unstimulated or stimulated with anti-CD3/CD28 for the indicated time points. Percentages of proliferated cells were determined by flow cytometry. Statistical analysis of N = 3–5, 5 independently performed experiments. The calculated power is 0.998 for day 2 and 1 for the rest of the measurements. (B) CD4 + T cells were stimulated with anti-CD3/CD28 without (control) or with SAS and erastin, and percentages of Ki-67+ cells were determined by flow cytometry. Statistical analysis of Ki-67+ cells with N = 4, 4 independently performed experiments. The calculated power of this experiment is 0.99. (C–F) CD4 + T cells were stimulated with anti-CD3/CD28 without (control) or with SAS or erastin, and percentages of IFN-γ+ (C), IL17+ (D), IL4+ (E), and HELIOS+Foxp3+ (F) T cells were determined by flow cytometry. Statistical analysis was performed with N = 3, 3 independently performed experiments, and N = 4, 4 independently performed experiments (E). For A-F, statistical analysis was performed using One-way ANOVA multiple comparison. The calculated power of the experiment is 0.83 (C), 0.99 (D), 0.11 (E), 0.99 (F). Data are presented as mean, error bars present ± SEM. Values were considered significant if ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. N represents the number of biological replicates.

Article Snippet: SLC7A11 , F: 5′ TGA CTG GAGTCC CTG CGT AT 3′; R: 5′ TGT TCT GGT TAT TTT CTC CGA CA 3′ , eurofins.

Techniques: Inhibition, Labeling, Flow Cytometry, Control, Comparison

Journal: iScience

Article Title: Inflammatory CD4 + T cells can waive NRF2-dependent SLC7A11-mediated cystine uptake by using ASCT1

doi: 10.1016/j.isci.2026.115680

Figure Lengend Snippet: Characterization of an altered NRF2/cystine pathway in patients with JIA (A) Flow cytometric analysis of SLC7A11 expression in stimulated SF and PB CD4 + T cells. Statistical analysis was performed with N = 7 donors, 7 independently performed experiments. (B) Flow cytometric evaluation of cystine uptake (shown as MFI) by anti-CD3/CD28 stimulated CD4 + SF T cells. N = 6, 6 independently performed experiments. (C) Flow cytometric analysis of intracellular glutathione content (presented as ΔMFI) of stimulated SF and PB CD4 + T cells. N = 9, 9 independently performed experiments. (D) Statistical analysis of MFI of ROS in stimulated SF and PB CD4 + T cells ( N = 4), 4 independently performed experiments. The calculated power of this experiment is 0.85. (E) Lipid peroxidation as assessed by flow cytometric measurement of anti-CD3/CD28 stimulated CD4 + T cells derived from PBMCs and SFMCs. N = 11. (F) Gene set enrichment analysis (GSEA) was performed on differentially expressed genes from SF-derived CD4 + T cells of patients with active JIA compared to PBMC-derived CD4 + T cells from healthy controls. The enrichment plots shown represent driver ferroptosis-related gene sets from KEGG, WikiPathways, and FerrDb V2. ( N = 4). (G) Heat maps showing normalized counts of selected RNAs determined by RNA-seq in JIA CD4 + T cells compared to HC CD4 + T cells. N = 4 in each group. (H) SFMCs and PBMCs were analyzed for ASCT1 expression by quantitative RT-PCR. The calculated power of this experiment is 0.99. (I) Flow cytometric analysis of CD36 expression of stimulated SF and PB CD4 + T cells. N = 5, 5 independently performed experiments. (J) Representative histograms showing BODIPY Ferroptosis staining of anti-CD3/CD28 stimulated HC PBMCs in the absence and presence of ASCT1/2 inhibition by 4-Hydroxy-L-phenylglycin (HPG) and SAS. N = 3, 3 independent experiments. The statistical evaluation of (A), (C), (E), (H), and (I) from the represented graphs was performed using a paired Student’s t-test. One-way ANOVA multiple comparison was used for (B) and (D) of the represented graphs. Data are presented as mean, error bars present ± SEM for all the presented graphs in this figure. Values were considered significant if ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. N represents the number of biological replicates.

Article Snippet: SLC7A11 , F: 5′ TGA CTG GAGTCC CTG CGT AT 3′; R: 5′ TGT TCT GGT TAT TTT CTC CGA CA 3′ , eurofins.

Techniques: Expressing, Derivative Assay, RNA Sequencing, Quantitative RT-PCR, Staining, Inhibition, Comparison

Journal: Frontiers in Pharmacology

Article Title: Apocynum venetum L. leaves extract inhibits ferroptosis and improves renal fibrosis in diabetic kidney disease by regulating the Nrf2/HO-1 pathway

doi: 10.3389/fphar.2026.1807373

Figure Lengend Snippet: AVLE inhibits ferroptosis in db/db mice. (A) TEM result. Scale bar = 2 or 0.5 μm, n = 3. (B,C) Identification of GPX4 protein expression in kidney tissues by IHC. Scale bar = 200 μm, n = 3. (D) Ferrous iron levels in kidney tissues of each group, n = 6. (E–G) Western blot for GPX4 and SLC7A11 protein expression with semi-quantitative analysis. β-actin served as the internal control. All data are presented as mean ± SEM (n = 6). Compared with the CON group: # p < 0.05, ## p < 0.01, ### p < 0.001; Compared with the MOD group: * p < 0.05, ** p < 0.01.

Article Snippet: SLC7A11 rabbit monoclonal antibody , 1:1000 , HUABIO , HA721868.

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Apocynum venetum L. leaves extract inhibits ferroptosis and improves renal fibrosis in diabetic kidney disease by regulating the Nrf2/HO-1 pathway

doi: 10.3389/fphar.2026.1807373

Figure Lengend Snippet: Impact of AVLE on Nrf2, HO-1 protein expression, and ferroptosis in HK-2 cells treated with HG. (A) Each group’s ferrous iron concentration was determined using a kit. (B,C) Each group’s levels of GPX4, SLC7A11, Nrf2, and HO-1 protein expression were identified by Western blot. All data are presented as mean ± SEM (n = 3). Compared with the NG group: # p < 0.05, ## p < 0.01, ### p < 0.001; compared with the HG group: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: SLC7A11 rabbit monoclonal antibody , 1:1000 , HUABIO , HA721868.

Techniques: Expressing, Concentration Assay, Western Blot