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