Journal: Redox Biology

Article Title: USP20 governs tyrosine kinase inhibitors resistance through ferroptosis evasion by targeting GPX4 in cancers

doi: 10.1016/j.redox.2026.104086

Figure Lengend Snippet: TKI-resistant cancer cells exhibit reduced ferroptosis susceptibility due to increased GPX4 stability. ( A ) Development of sorafenib-resistant (Sora-R) cell models. Renal carcinoma 769-P cells were subjected to chronic sorafenib exposure (initial 0.5 μM for 5 days, followed by 1 μM dose escalation every 5 days until reaching 10 μM, then cultured in medium with 10 μM sorafenib for 3 month). Surviving populations were designated as Sora-R. The validity of this treatment was assessed in parental and Sora-R cells after 24 h sorafenib treatment using MTT assays. ( B ) The validity of sorafenib-resistant cells with colony formation assay. Sorafenib-resistant and parental cells was cultured for 7 days while treated with indicated concentration of sorafenib. The colony number was measured with Image J. ( C-D ) Parental and Sora-R 769-P cells were treated with 30 μM sorafenib for 6 h after pretreated with Tempol (50 μM), EUK-143 (50 μM), DFO (50 μM), and Fer-1 (1 μM) for 2 h, followed by quantification of total ROS using fluorescence microscope with DCFH-DA probe. Scale bar: 50 μm. ( E ) ROS generation and clearance. ( F ) Parental and Sora-R 769-P cells were treated with 30 μM sorafenib for 6 h, then quantified of GPX4 activity. ( G-H ) Parental and Sora-R 769-P cells were treated with 30 μM sorafenib for 6 h after pretreated with Tempol (50 μM), EUK-143 (50 μM), DFO (50 μM), and Fer-1 (1 μM) for 2 h, followed by quantification of lipid ROS using fluorescence microscope with C11-BODIPY 581/591 probe. Scale bar: 50 μm. ( I ) Parental and Sora-R 769-P cells were treated with 30 μM sorafenib for 6 h after pretreated with DFO (50 μM) and Fer-1 (1 μM) for 2 h, followed by quantification of MDA via ELISA kits. ( J-K ) Parental and Sora-R cells were treated with 30 μM IKE for 6 h, followed by quantification of lipid ROS using fluorescence microscope with C11-BODIPY 581/591 probe. Scale bar: 50 μm. ( L ) 769-P cells were treated with 20 μM sorafenib for 24 h after pretreated with 1 μM Fer-1, 2 μM Lip-1, 50 μM DFO, 10 μM Nec-1, 25 μM Z-VAD-FMK, 25 μM 3-MA, 50 μM CQ, and 10 μM TTM, cell viability was measured via MTT assay. ( M-N ) Immunoblot analysis of key antioxidant and ferroptosis-related proteins in parental and Sora-R cell lines. For A-D and F-L , data are presented as mean ± s.d. of n = 3 biological replicates.

Article Snippet: Cells were seeded in 6-well plates and, upon reaching appropriate confluence, incubated with 10 μM DCFH-DA (MedChemExpress, Cat. No. HY-15933) in serum-free medium at 37 °C for 30 min. After washing twice with PBS, cells were counterstained with 5 μg/mL Hoechst 33342 (MedChemExpress, Cat. No. HY-15559) for 10 min to visualize nuclei.

Techniques: Cell Culture, Colony Assay, Concentration Assay, Fluorescence, Microscopy, Activity Assay, Enzyme-linked Immunosorbent Assay, MTT Assay, Western Blot