hek 293t cell line  (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 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: SMURF2 attenuates NRF2-driven tumor progression by acting as a nuclear brake on NRF2 during cellular stress

doi: 10.1016/j.redox.2026.104102

Figure Lengend Snippet: SMURF2 prohibits the stress-mediated formation of ub + /p62 + aggresomes. (A and B) LN229 cells were transfected with HA or HA-SMURF2 and treated with DMSO; MG132 (10 μM, 12 h); H 2 O 2 (200 μM, 2 h); or LPS (100 ng/mL, 12 h). Representative immunofluorescence (IF) images of the colocalization of ub and p62. Nuclei stained with DAPI (A). Quantification the percentage of cells with ub + /p62 + puncta (B). (C – E) HEK293T cells were transfected with HA or HA-SMURF2 and subsequently treated with MG132 (10 μM, 12 h) or H 2 O 2 (200 μM, 2 h). Detergent-soluble and detergent-insoluble fractions were analyzed by western blotting with indicated antibodies (C and D). Quantification of the relative intensity of ub and p62 levels in the detergent-insoluble fractions shown in C and D (E). (F) LN229 cells were transfected with HA or HA-SMURF2 and treated with DMSO; MG132 (10 μM, 12 h); H 2 O 2 (200 μM, 2 h); or LPS (100 ng/mL, 12 h). Representative IF images of the colocalization of Proteostat and p62. Nuclei stained with DAPI. (G) The proposed model suggests that SMURF2 inhibits the formation of ub + /p62 + aggresomes/ALIS under stress conditions. Data were presented as the mean ± SD from three independent experiments. NS: not significant, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Scale bar: 10 μm. Short Exp: short exposure; Long Exp: long exposure.

Article Snippet: The human glioblastoma cell lines LN229 and human embryonic kidney 293T (HEK293T) cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Immunofluorescence, Staining, Western Blot

Journal: Redox Biology

Article Title: SMURF2 attenuates NRF2-driven tumor progression by acting as a nuclear brake on NRF2 during cellular stress

doi: 10.1016/j.redox.2026.104102

Figure Lengend Snippet: SMURF2 promotes NRF2 proteasomal degradation in response to cellular stress. (A) Co-immunoprecipitation (Co-IP) assay was performed to analyze the interaction between SMURF2 and NRF2. (B) Co-IP assay analysis of the interaction between HA-SMURF2 and His-Flag-NRF2 after treated with or without H 2 O 2 (200 μM, 2 h) (C) Endogenous co-IP assay analysis of the interaction between endogenous SMURF2 and NRF2 in HEK293T cells after treated with or without H 2 O 2 (200 μM, 2 h). (D and E) Co-IP assay analysis of the interaction between His-Flag-SMURF2 constructs (WT, C2, WW3 and ΔHECT) and Myc-NRF2 (D); the interaction between GST-SMURF2 constructs (ΔC2 and ΔWW3) and HA-NRF2 (E). (F) Co-IP assay analysis of the interaction between His-Flag-NRF2 constructs (WT and ΔNeh1-6) and HA-SMURF2. (G) Schematic diagram of mapping the direct interaction between SMURF2 and NRF2. (H) HEK293T cells expressing Flag-NRF2 were treated with MG132 (10 μM,12 h). The ubiquitination of Flag-NRF2 in the presence of purified GST or GST-SMURF2 was then detected by western blotting. (I) HEK293T cells were transfected with SMURF2 siRNA or scramble siRNA for 48 h, then transfected with HA-NRF2 and restored with Flag-SMURF2-WT/CS/CS C716A , treated with MG132 (10 μM, 12 h). Ubiquitination of HA-NRF2 was assessed by co-IP after SMURF2 knockdown and functional restoration. (J) HEK293T cells expressing HA-NRF2 and Flag-Ub-K48 or Flag-Ub-K63. The K48-linked or K63-linked ubiquitination of HA-NRF2 in the presence of purified GST or GST-SMURF2 was then detected by western blotting. (K) HEK293T cells were transfected with either HA or HA-SMURF2, then treated with DMSO, MG132 (10 μM, 12 h) or Bafilomycin A1 (Baf-A1, 100 nM, 6 h) and analyzed by western blotting of whole cell lysates (WCL) using the indicated antibodies. (L – O) HEK293T cells were transfected either with HA or HA-SMURF2 (L), or with SMURF2 siRNA or scramble siRNA oligos for 48 h (N), then treated with cycloheximide (CHX, 100 μg/mL) for the indicated times and analyzed by western blotting using the indicated antibodies. Quantification of the relative intensity of NRF2 is shown (M, O). (P) HEK293T cells were transfected with either Flag or Flag-SMURF2, then treated with PBS, H 2 O 2 (200 μM, 2 h), or LPS (100 ng/mL, 12 h) and analyzed by western blotting using the indicated antibodies. (Q) HEK293T cells were transfected with either HA or HA-SMURF2, then treated with PBS or LPS (100 ng/mL, 12 h) and analyzed by qRT-PCR using primers specific for indicated genes. The fold change in expression in HA-SMURF2 overexpressing samples was calculated relative to control samples. (R) HEK293T cells were transfected with SMURF2 siRNA or scramble siRNA oligos for 48 h, then treated with PBS, H 2 O 2 (200 μM, 2 h), or LPS (100 ng/mL, 12 h) and analyzed by western blotting using the indicated antibodies. (S) HEK293T cells were transfected with SMURF2 siRNA or scramble siRNA oligos for 48 h, then treated with PBS or LPS (100 ng/mL, 12 h) and analyzed by qRT-PCR using primers specific for indicated genes. The fold change in expression in si-SMURF2 samples was calculated relative to control samples. Data were presented as the mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The human glioblastoma cell lines LN229 and human embryonic kidney 293T (HEK293T) cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Co-Immunoprecipitation Assay, Construct, Expressing, Ubiquitin Proteomics, Purification, Western Blot, Transfection, Knockdown, Functional Assay, Quantitative RT-PCR, Control

Journal: Redox Biology

Article Title: SMURF2 attenuates NRF2-driven tumor progression by acting as a nuclear brake on NRF2 during cellular stress

doi: 10.1016/j.redox.2026.104102

Figure Lengend Snippet: SMURF2 nuclear translocation facilitates the degradation of NRF2 within the nucleus. (A and B) HEK293T cells were first transfected with HA-NRF2-WT. Subsequently, cells were transfected either with Flag or Flag-SMURF2 (A), or with SMURF2 siRNA or scrambled siRNA oligos for 48 h (B). Cells were then treated with H 2 O 2 (200 μM, 2 h) or LPS (100 ng/mL, 12 h). Following treatment, subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. (C and D) LN229 cells were transfected with HA-SMURF2, then treated with PBS, H 2 O 2 (200 μM, 2 h) (C) or LPS (100 ng/mL, 12 h) (D). Representative IF images showing the subcellular localization of HA-SMURF2 are presented; nuclei were stained with DAPI. (E and F) HEK293T cells were treated with PBS, H 2 O 2 (200 μM, 2 h) (E), or LPS (100 ng/mL, 12 h) (F). Subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. (G) Schematic depiction of NRF2 NLS1/2−Mut , NRF2 NES1/2−Mut , and SMURF2 NLS−Mut . (H–K) HEK293T cells were first transfected with HA-NRF2 NLS2−Mut (H and I) or HA-NRF2 NES2−Mut (J and K). Subsequently, cells were transfected either with Flag or Flag-SMURF2, then treated with H 2 O 2 (200 μM, 2 h) (H and J) or LPS (100 ng/mL, 12 h) (I and K). Following treatment, subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. (L) Schematic of SMURF2 domains with NLS indicated (single-letter code). SMURF2 NLS−Mut denotes deletion of NLS residues (Top). LN229 cells transfected with Flag-SMURF2-WT or Flag-SMURF2 NLS−Mut were treated with DMSO, MG132 (10 μM, 12 h), H 2 O 2 (200 μM, 2 h), or LPS (100 ng/mL, 12 h). Representative IF images showing subcellular localization of Flag-SMURF2 or Flag-SMURF2 NLS−Mut ; nuclei were stained with DAPI (Bottom). (M) HEK293T cells were first transfected with HA-NRF2-WT. Subsequently, cells were transfected either with Flag or Flag-SMURF2 NLS−Mut , then treated with H 2 O 2 (200 μM, 2 h) or LPS (100 ng/mL, 12 h). Following treatment, subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. (N) The proposed model indicates that SMURF2 specifically degrades NRF2 in the nucleus. Scale bar: 5 μm, Scale bar: 10 μm. Data were presented in three independent experiments.

Article Snippet: The human glioblastoma cell lines LN229 and human embryonic kidney 293T (HEK293T) cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Translocation Assay, Transfection, Fractionation, Western Blot, Staining

Journal: Redox Biology

Article Title: SMURF2 attenuates NRF2-driven tumor progression by acting as a nuclear brake on NRF2 during cellular stress

doi: 10.1016/j.redox.2026.104102

Figure Lengend Snippet: SMURF2 ubiquitinates NRF2 at K555 in the nucleus for its degradation. (A) HEK293T cells were transfected with HA-NRF2 constructs (ΔNeh1-ΔNeh6), followed by treatment with MG132 (10 μM, 12 h). The ubiquitination of HA-NRF2 constructs (ΔNeh1-ΔNeh6) in the presence of purified GST or GST-SMURF2 was then detected by western blotting. (B) HEK293T cells were transfected with HA-NRF2 constructs (Δ434-500, Δ501-539 and Δ540-561), followed by treatment with MG132 (10 μM, 12 h). The ubiquitination of HA-NRF2 constructs (Δ434-500, Δ501-539 and Δ540-561) in the presence of purified GST or GST-SMURF2 was then detected by western blotting. (C) HEK293T cells were transfected with HA-NRF2 constructs (K554R/K555R and K541R/K543R/K548R), followed by treatment with MG132 (10 μM, 12 h). The ubiquitination of HA-NRF2 constructs (K554R/K555R and K541R/K543R/K548R) in the presence of purified GST or GST-SMURF2 was then detected by western blotting. (D) HEK293T cells were transfected with HA-NRF2 constructs (K554R and K555R), followed by treatment with MG132 (10 μM, 12 h). The ubiquitination of HA-NRF2 constructs (K554R and K555R) in the presence of purified GST or GST-SMURF2 was then detected by western blotting. (E) Purified SMURF2 with His-ub, E1, E2 (UbcH5c), and ATP were used as indicated and evaluated by pull-down assay. Ubiquitinated NRF2-WT and K555R detected by immunoblotting against anti-Ub and anti-Flag. (F) Sequence alignment of NRF2 sites on CNA orthologs of different species. (G) HEK293T cells overexpressing HA-NRF2 were transfected with Flag or Flag-SMURF2, followed by treatment with MG132 (10 μM, 12 h). Following treatment, subcellular fractionation was performed to isolate cytoplasmic and nuclear fractions. The ubiquitination of cytoplasmic and nuclear fractions was then detected by western blotting. (H) HEK293T cells were transfected with HA-NRF2 NLS1−Mut , HA-NRF2 NES1−Mut , HA-NRF2 NLS2−Mut or HA-NRF2 NES2−Mut , followed by treatment with MG132 (10 μM, 12 h). The ubiquitination of HA-NRF2 constructs in the presence of purified GST or GST-SMURF2 was then detected by western blotting. (I and J) HEK293T cells were first transfected with HA-NRF2 (I), or HA-NRF2-K555R (J), then were transfected with Flag or Flag-SMURF2, followed by treatment with MG132 (10 μM, 12 h) (I), H 2 O 2 (200 μM, 2 h) or LPS (100 ng/mL, 12 h) (J). Following treatment, subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. Data were presented in three independent experiments.

Article Snippet: The human glioblastoma cell lines LN229 and human embryonic kidney 293T (HEK293T) cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Construct, Ubiquitin Proteomics, Purification, Western Blot, Pull Down Assay, Sequencing, Fractionation

Journal: Redox Biology

Article Title: SMURF2 attenuates NRF2-driven tumor progression by acting as a nuclear brake on NRF2 during cellular stress

doi: 10.1016/j.redox.2026.104102

Figure Lengend Snippet: SMURF2 promotes cell apoptosis through NRF2 inactivation. (A) LN229 cells were transfected with HA, HA-SMURF2, Myc-NRF2 or HA-SMURF2 + Myc-NRF2 and subsequent treatment with MG132 (10 μM, 12 h), H 2 O 2 (200 μM, 2 h), or LPS (100 ng/mL, 12 h). Representative IF images of the colocalization of ub and p62. Nuclei stained with DAPI. (B and C) HEK293T cells were transfected with empty vector or Myc-NRF2, followed by Flag or Flag-SMURF2 expression. Western blotting was performed using indicated antibodies (B). qRT-PCR was performed using primers specific for indicated genes (C). The fold change in expression in Flag-SMURF2 overexpressing samples was calculated relative to control samples. (D) HEK293T cells were first transfected with NRF2 siRNA for 48 h, followed by transfection with either Myc-NRF2-WT or Myc-NRF2-K555R, and subsequently transfected with Flag or Flag-SMURF2.Western blotting was performed using indicated antibodies. (E) LN229 cells were transfected with SMURF2 siRNA or scramble siRNA oligos for 48 h, followed by treatment with PBS or H 2 O 2 (200 μM, 2 h). Cells were then stained with 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) and the ROS level was detected by flow cytometry. (F) Quantification of relative ROS fluorescence intensity in LN229 cells with SMURF2 knockdown or HA-SMURF2 overexpression. (G and H) LN229 cells were transfected with SMURF2 siRNA or scramble siRNA oligos for 48 h or transfected with HA or HA-SMURF2, followed by treatment with PBS or H 2 O 2 (200 μM, 2 h). Cells were then stained with Annexin-V/propidium iodide (PI), and apoptotic cells were detected by flow cytometry (G). Quantification of apoptosis in LN229 cells with SMURF2 knockdown or HA-SMURF2 overexpression (H). (I) HEK293T cells were transfection with Flag or Flag-SMURF2, and subsequent treatment with or without MG132 (10 μM, 12 h), H 2 O 2 (200 μM, 2 h), or LPS (100 ng/ml, 12 h). Western blotting was performed using indicated antibodies. (J and K) HEK293T cells were transfected with empty vector and Myc-NRF2 (J) or Myc-NRF2-WT, Myc-NRF2-K555R (K), followed by Flag or Flag-SMURF2 expression. Cells were subsequently treated with MG132 (10 μM, 12 h), H 2 O 2 (200 μM, 2 h), or LPS (100 ng/mL, 12 h). Western blotting was performed using indicated antibodies. (L) The shSMURF2 and shPLKO cells were treated with or without LPS (100 ng/mL, 12 h) and cultured for 14 days. Colony formation assay was performed for cells. (M) The representative images of shSMURF2 and shPLKO patient-derived cells formed tumors in nude mice with or without LPS (10 mg/kg). (N) The graph showed the quantified data of tumor weight. (O) Immunohistochemistry (IHC) analysis of tumor tissue slides with antibodies against Ki67. Nucleus was stained by hematoxylin. Scale bar, 50 μm (P) The proposed model indicates that SMURF2 promotes cell apoptosis through NRF2 inactivation. Data were presented as the mean ± SD from three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Scale bar: 10 μm, Scale bar, 50 μm.

Article Snippet: The human glioblastoma cell lines LN229 and human embryonic kidney 293T (HEK293T) cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Staining, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR, Control, Flow Cytometry, Fluorescence, Knockdown, Over Expression, Cell Culture, Colony Assay, Derivative Assay, Immunohistochemistry

Journal: Redox Biology

Article Title: SMURF2 attenuates NRF2-driven tumor progression by acting as a nuclear brake on NRF2 during cellular stress

doi: 10.1016/j.redox.2026.104102

Figure Lengend Snippet: SMURF2 promotes NRF2 hi patient survival. (A) HA-NRF2 overexpressing HEK293T cells were transfected with GFP or GFP-KEAP1, followed by transfection with Flag or Flag-SMURF2. Co-IP analysis of the interaction between HA-NRF2 and GFP-KEAP1 in WCL. (B) Myc-NRF2 overexpressing HEK293T cells were transfected with HA or HA-KEAP1, followed by transfection with Flag or Flag-SMURF2. Co-IP analysis of the interaction between Myc-NRF2 and HA-KEAP1 in the cytoplasm. (C) HA-NRF2 overexpressing HEK293T cells were transfected with GFP or GFP-KEAP1, followed by transfection with SMURF2 siRNA or scramble siRNA oligos for 48 h. Co-IP analysis of the interaction between HA-NRF2 and GFP-KEAP1 in the cytoplasm under with or without SMURF2. (D) HEK293T cells were initially transfected with KEAP1 siRNA for 48 h, followed by transfection with HA-NRF2-WT, and subsequently transfected with Flag or Flag-SMURF2. Subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. (E) HEK293T cells were first transfected with HA-NRF2-WT. Subsequently, cells were transfected either with Flag or Flag-KEAP1. Subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. (F) GFP-KEAP1 overexpression HEK293T cells were transfected with HA-NRF2-WT, and subsequently transfected with Flag or Flag-SMURF2. Subcellular fractionation was performed to isolate total, nuclear, and cytoplasmic proteins, followed by western blot analysis with indicated antibodies. (G) Co-IP assay analysis of the interaction between HA-KEAP1 and His-Flag-NRF2 after treated with or without H 2 O 2 (200 μM, 2 h) (H) Boxplot of SMURF2 expression (log 2 FPKM) in the NRF2-high/KEAP1-low subset of TCGA-GBMLGG samples. (I) Kaplan-Meier survival analysis of TCGA-GBMLGG patients grouped by expression of SMURF2 in the NRF2-high/KEAP1-low subset ( p = 0.018, HR = 1.7755, log rank test). (J) Clinical feature distribution across SMURF2 expression groups. (K) Forest Plot of Multivariable Cox Proportional Hazards Analysis in High-Grade Gliomas. Data were presented in three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: The human glioblastoma cell lines LN229 and human embryonic kidney 293T (HEK293T) cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Co-Immunoprecipitation Assay, Fractionation, Western Blot, Over Expression, Expressing