nsclc cell lines h1299  (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: 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: bioRxiv

Article Title: Dual inhibition of GTP-bound (ON) and GDP-bound (OFF) KRAS G12C suppresses PI3Kα and leads to potent tumor inhibition

doi: 10.64898/2026.04.27.718135

Figure Lengend Snippet: a, Composite dose response curves of KRAS G12C -mutant NSCLC cell lines. Cells were treated with KRAS inhibitors for 72 hours and cell viability was quantified by CellTiter-Glo. Curves shown are mean and S.E.M. of n=3-7 independent biological replicates. b-c, Comparison of IC50 values from 3-day viability assays. Values shown are mean and S.E.M. of n=3-7 independent biological replicates. d-e, Comparison of Emax values from 3-day viability assays. Values shown are mean and S.E.M. of n=3-7 biological replicates. f, Western blot analysis of KRAS G12C -mutant NSCLC cell lines treated with increasing concentrations of sotorasib or BBO-8520 for 6 hours. Data is representative of n=3 independent biological replicates.

Article Snippet: All in vivo procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) prior to execution and performed in accordance with the regulations and guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care at MGH, NYU, Champions Oncology, Inc., or Crown Biosciences, Inc. KRAS -mutant NSCLC PDX models were generated by subcutaneous implantation of tumor cells or tissue from malignant pleural effusions, core-needle biopsies, or surgical resections into male NSG mice (Jackson Labs).

Techniques: Mutagenesis, Comparison, Western Blot

Journal: bioRxiv

Article Title: Dual inhibition of GTP-bound (ON) and GDP-bound (OFF) KRAS G12C suppresses PI3Kα and leads to potent tumor inhibition

doi: 10.64898/2026.04.27.718135

Figure Lengend Snippet: a, Imaged-based monitoring (Incucyte) of proliferation of KRAS G12C -mutant NSCLC cell lines treated with sotorasib or BBO-8520. Data are mean and S.E.M of n=4-6 technical replicates and are representative of n=3 independent biological replicates. b, Relative cell proliferation of cells treated with BBO-8520, normalized to sotorasib. Values are mean of n=3 biological replicates, corresponding to the cell counts shown in panel A. c, Comparison of cell proliferation after treatment with 10 nM, 100 nM or 1 μM BBO-8520 or 1 μM sotorasib. Values represent cell proliferation relative to baseline determined after 10 days from three independent biological replicates. d, Relative cell proliferation of cells treated with BBO-8520, normalized to sotorasib after 10 days (H2122, MGH1114-1, MGH1062-1, H23, H2030), 6 days (H1792), or 5 days (HCC44) to account for variability in baseline (vehicle-treated) growth rates. Values are mean of n=3-4 biological replicates.

Article Snippet: All in vivo procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) prior to execution and performed in accordance with the regulations and guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care at MGH, NYU, Champions Oncology, Inc., or Crown Biosciences, Inc. KRAS -mutant NSCLC PDX models were generated by subcutaneous implantation of tumor cells or tissue from malignant pleural effusions, core-needle biopsies, or surgical resections into male NSG mice (Jackson Labs).

Techniques: Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Dual inhibition of GTP-bound (ON) and GDP-bound (OFF) KRAS G12C suppresses PI3Kα and leads to potent tumor inhibition

doi: 10.64898/2026.04.27.718135

Figure Lengend Snippet: a, KRAS G12C -mutant NSCLC cells were treated with BBO-8520 (100 nM) or sotorasib (1 μM) for 6 or 48 hours and RAF-RBD pull-down was performed to assess engagement between KRAS-GTP and RAF. Data are quantified band intensities from western blots (see Supplemental Figure 3a) normalized to vehicle control, n=3 independent biological replicates. b, Average change in KRAS-GTP levels after 6 or 48 hours determined by RAF-RBD pull-down. Each data point represents the average change in KRAS-GTP in an individual cell line from panel A. c, KRAS G12C -mutant NSCLC cells were treated with BBO-8520 (100 nM) or sotorasib (1 μM) for 2 weeks and RAF-RBD pull-down was performed to assess engagement between KRAS-GTP and RAF in residual tumor cells. Data are quantified band intensities from western blots normalized to vehicle control, n=4-6 independent biological replicates. d, Average change in KRAS-GTP levels after 2 weeks as determined by RAF-RBD pull-down. Each data point represents the average change in KRAS-GTP in an individual cell line from panel D. e, Representative western blot images from RAF-RBD pull-down after 2 weeks treatment with sotorasib or BBO-8520, as quantified in panels C and D. f-g, Average pERK (F) and pAKT (G) levels after treatment with BBO-8520 (100 nM) or sotorasib (1 μM) determined by western blot band quantification (see panel E). Each dot represents the mean of an individual cell line, n=4-6 independent biological replicates.

Article Snippet: All in vivo procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) prior to execution and performed in accordance with the regulations and guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care at MGH, NYU, Champions Oncology, Inc., or Crown Biosciences, Inc. KRAS -mutant NSCLC PDX models were generated by subcutaneous implantation of tumor cells or tissue from malignant pleural effusions, core-needle biopsies, or surgical resections into male NSG mice (Jackson Labs).

Techniques: Mutagenesis, Western Blot, Control