human rcc cell lines 769 p  (ATCC)

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: Human RCC cell lines 769-P, 786-O and SW839, human NSCLC cell lines A549, H1299 and the HEK-293T cell line were obtained from the American Type Culture Collection and cultured in RPMI-1640 or DMEM medium (Thermo Fisher Scientific, Inc.) added with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.),100 U/ml penicillin and 0.1 mg/ml streptomycin (Thermo Fisher Scientific, Inc.) at a temperature of 37 °C.

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

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: USP20 directly interacts with GPX4. ( A ) Immunoblot analysis of target proteins in sorafenib-resistant 769-P and A549 cells following 24 h treatment with specific deubiquitinase inhibitors. ML-323: 10 μM, GSK2643943A: 10 μM, XL177A: 10 μM, Spautin-1: 10 μM, IU1: 10 μM, AZ1: 10 μM, MF-094: 10 μM, LDN-57444: 10 μM, TCID: 10 μM, PR-619: 10 μM, BAY11-7082: 10 μM, EOAI3402143: 2.5 μM, ML364: 10 μM, b-AP15: 0.5 μM. ( B ) HEK293T cells were transfected with GST-GPX4 and Flag-USP. The cell lysate was applied with GST pull-down, then analyzed with immunoblot for indicated proteins. ( C ) 769-P and A549 cells were subjected to immunoprecipitate with anti-GPX4 antibodies, then analyzed with immunoblot for indicated proteins. ( D ) Overview of USP20 structures. ( E-F ) HEK293T cells transfected with the indicated USP20 structures and Flag-GPX4 were subjected to pull-down with GSH beads or immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins. ( G ) 769-P and A549 cells were subjected to subcellular fractionation, then analyzed with immunoblot for indicated proteins. ( H ) Overview of GPX4 isoforms structures. ( I ) HEK293T cells transfected with the indicated GPX4 isoforms and GST-USP20 were subjected to immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins. ( J ) HEK293T cells transfected with the GST-USP20 WT/C154S and Flag-GPX4 were subjected to immunoprecipitate with anti-Flag antibody, then analyzed with immunoblot for indicated proteins.

Article Snippet: Human RCC cell lines 769-P, 786-O and SW839, human NSCLC cell lines A549, H1299 and the HEK-293T cell line were obtained from the American Type Culture Collection and cultured in RPMI-1640 or DMEM medium (Thermo Fisher Scientific, Inc.) added with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.),100 U/ml penicillin and 0.1 mg/ml streptomycin (Thermo Fisher Scientific, Inc.) at a temperature of 37 °C.

Techniques: Western Blot, Transfection, Fractionation

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: USP20 elevates GPX4 protein abundance through post-translational regulation. ( A ) Western blot analysis of GPX4 and USP20 proteins in 769-P and A549 cells transfected with lentivirus carrying shUSP20 or negative control. ( B ) Western blot analysis of GPX4 and USP20 proteins in 786-O and HEK 293T cells transfected with GST-USP20 plasmids or vector. ( C-D ) Analysis of GPX4 and USP20 mRNA expression in shScr and shUSP20 769-P and A549 cells. ( E-H ) shScr and shUSP20 769-P (E-F) and A549 (G-H) cells were treated with 100 μg/mL CHX at indicated time points. The GPX4 protein abundance was quantified by the Image J and statistical charts are produced by GraphPad. ( I-J ) shScr and shUSP20 769-P and A549 cells were treated with 20 μM MG132 for 6 h or 20 μM CQ for 12 h, and then assessed GPX4 expression. ( K ) Western blot analysis of GPX4 proteins in HEK293 cells transfected with GST-USP20 WT or GST-USP20 C154S plasmids. ( L-M ) Western blot analysis of GPX4 proteins in USP20 knockdown HEK293 cells transfected with GST-Vector, GST-USP20 WT or GST-USP20 C154S plasmids. The GPX4 protein abundance was quantified by the Image J software and statistical charts are produced by GraphPad. ( N ) Western blot analysis of GPX4 proteins in 769-P, SW839, A549, H1299 cells treated with indicated concentration GSK2643943A for 24 h. ( O–R ) The 769-P and A549 cells were treated with 10 μM GSK2643943A for 24 h, then 100 μg/mL CHX at indicated time points. The GPX4 protein abundance was quantified by the Image J software and statistical charts are produced by GraphPad. Data in C and D are presented as mean ± s.d. of n = 3 biological replicates.

Article Snippet: Human RCC cell lines 769-P, 786-O and SW839, human NSCLC cell lines A549, H1299 and the HEK-293T cell line were obtained from the American Type Culture Collection and cultured in RPMI-1640 or DMEM medium (Thermo Fisher Scientific, Inc.) added with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.),100 U/ml penicillin and 0.1 mg/ml streptomycin (Thermo Fisher Scientific, Inc.) at a temperature of 37 °C.

Techniques: Quantitative Proteomics, Western Blot, Transfection, Negative Control, Plasmid Preparation, Expressing, Produced, Knockdown, Software, Concentration Assay

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: USP20 deficiency sensitizes cancer cells to FINs. ( A-B ) shScr and shUSP20 769-P (A) and A549 (B) cells were treated with 20 μM IKE for 6 h, followed by quantification of lipid ROS with C11-BODIPY 581/591 probe. ( C-D ) shScr and shUSP20 769-P (C) and A549 (D) cells were treated with indicated concentration of IKE and 1 μM Fer-1 for 48 h Cell viability was measured via MTT assay. ( E ) 786-O cells, transfected with EV or USP20-overexpressing plasmid, were treated with indicated concentration of IKE and 1 μM Fer-1 for 48 h Cell viability was measured via MTT assay. ( F-G ) shScr and shUSP20 tumor cells were cultured for 10 days while treated with 10 μM IKE and 1 μM Fer-1, the colony number was measured with Image J. ( H–I ) 786-O cells, transfected with EV or USP20-overexpressing plasmid, were cultured for 10 days while treated with 10 μM IKE and 1 μM Fer-1, the colony number was measured with Image J. ( J-N ) shScr and shUSP20 769-P cells were planted in nude mouse. After the xenografts reached 100 mm 3 , mice were treated with IKE (100 mg/kg) and Fer-1 (5 mg/kg) every three days, tumor volume (J) was measure at indicated times, xenografts were weighted at the day 32(K-L). The expressions of USP20, GPX4 and 4-HNE in the xenograft tumors were detected by immunohistochemistry and analyzed via IHC score (N). (Scale bar, 100 μM). ( O–P ) EV and USP20-overexpressing 786-O cells were planted in nude mouse. After the xenografts reached 100 mm 3 , mice were treated with IKE (100 mg/kg) and Fer-1 (5 mg/kg) every three days, tumor volume (O) was measure at indicated times, xenografts were weighted at the day 32 (P). For A-I , data are presented as mean ± s.d. of n = 3 biological replicates. Data in J and O is presented as mean ± s.e.m., data in K, L and N is presented as mean ± s.d. n = 5 mice. shScr: shScramble. EV: empty vector. OE: USP20-overexpressing.

Article Snippet: Human RCC cell lines 769-P, 786-O and SW839, human NSCLC cell lines A549, H1299 and the HEK-293T cell line were obtained from the American Type Culture Collection and cultured in RPMI-1640 or DMEM medium (Thermo Fisher Scientific, Inc.) added with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.),100 U/ml penicillin and 0.1 mg/ml streptomycin (Thermo Fisher Scientific, Inc.) at a temperature of 37 °C.

Techniques: Concentration Assay, MTT Assay, Transfection, Plasmid Preparation, Cell Culture, Immunohistochemistry

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: Inhibition of USP20 sensitives cancer cell to TKIs in vivo and in vitro . ( A ) Sorafenib-resistant 769-P cells were transfected with lentivirus expressing shRNA or shUSP20. Then cells were treated with indicated concentration of sorafenib for 24 h Cell viability was measured via MTT assay. ( B ) Sorafenib-resistant 769-P cells were treated with 10 μM GSK2643943A, following treated with indicated concentration of sorafenib for 24 h Cell viability was measured via MTT assay. ( C-D ) Sorafenib-resistant 769-P cells were treated with 10 μM GSK2643943A and 10 μM sorafenib for 10 days, the colony number was measured with Image J. ( E-J ) The growth curves of parental and sorafenib-resistant (Sora-R) 769-P xenografts in different treatment groups (GSK2643943A: 100 mg/kg every three days,7 times; Sorafenib: 100 mg/kg every three days, 7 times). Tumor volumes were measured every three days from day 14 to day 32 (E). After 32 days, the nude mice were sacrificed, and the xenografts were weighed (F-G). The expressions of USP20, GPX4 and 4-HNE in the xenograft tumors were measured by immunohistochemistry. (H, Scale bar, 100 μm). The IHC score of USP20 and 4-HNE of H (I-J). n = 5 mice per treatment group, data are presented as mean ± SD. ( K ) A model of USP20 controlling GPX4 protein at the post-translational level to ameliorate IKE-induced ferroptosis. In renal and lung carcinoma cells resistant to TKIs, the deubiquitinase USP20 is elevated. This upregulation stabilizes the GPX4 protein by removing its K48-linked polyubiquitin chains, thereby suppressing ferroptosis. Pharmacological inhibition of USP20 with GSK2643943A augments sorafenib-induced ferroptosis and restores tumor sensitivity to sorafenib. For A-D , data are presented as mean ± s.d. of n = 3 biological replicates; data in E is presented as mean ± s.e.m., data in G, I and J are presented as mean ± s.d. n = 5 mice.

Article Snippet: Human RCC cell lines 769-P, 786-O and SW839, human NSCLC cell lines A549, H1299 and the HEK-293T cell line were obtained from the American Type Culture Collection and cultured in RPMI-1640 or DMEM medium (Thermo Fisher Scientific, Inc.) added with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.),100 U/ml penicillin and 0.1 mg/ml streptomycin (Thermo Fisher Scientific, Inc.) at a temperature of 37 °C.

Techniques: Inhibition, In Vivo, In Vitro, Transfection, Expressing, shRNA, Concentration Assay, MTT Assay, Immunohistochemistry