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ATCC human embryonic kidney 293t hek293t cells
Human Embryonic Kidney 293t Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 38021 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 38021 article reviews

human embryonic kidney 293t hek293t cells - by Bioz Stars, 2026-09

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Cell Culture:

Article Title: Minigene-based splicing analysis uncovers pathogenic splice-altering effects of PAX2.
Article Snippet: .. Cell Culture and Transfection For minigene assays, human embryonic kidney 293T (HEK293T) cells were purchased from the American Type Culture Collection (ATCC, USA) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and incubated at 37 °C and 5% CO2. .. One day before transient transfection, the cells were seeded in 6-well cell culture plates, reached 70-80% confluency and transfected 4 μg plasmids per well using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Article Title: HCMV-pUS2 Disrupts cGAS-STING Signaling through LMAN2L Degradation
Article Snippet: .. Human embryonic kidney 293T (HEK293T) cells were purchased from ATCC (CRL-11268) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; cat. no. 41500–034; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; cat. no. SXRS-FBS-001; RUNSUN Biotech) and penicillin-streptomycin (100 U/ml and 100 μg/ml, respectively; cat. no. 15140–122; Thermo Fisher Scientific). ..

Article Title: Unraveling the role of ChREBP in lung adenocarcinoma: Expression, regulatory networks, and potential functional impact
Article Snippet: The HepG2 cell line was maintained in Eagle’s Minimal Essential Medium (ATCC, USA) with 10% FBS (Gibco, USA). .. Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco#39;s Modified Eagle Medium (ATCC, USA) supplemented with 10% FBS (Gibco, USA). ..

Transfection:

Modification:

Incubation:

Multiple Displacement Amplification:

Article Title: Structural and functional insights into an anticancer peptide candidate derived from the EP400NL C-terminal domain.
Article Snippet: EP400 N-terminal Like (EP400NL) is a recently characterized transcriptional coactivator implicated in chromatin remodeling and oncogenic signaling.. Although its precise function remains incompletely understood, emerging evidence suggests that EP400NL contributes to transcriptional regulatory networks relevant to tumor progression.. In this study, we investigated the structural and functional properties of the C-terminal domain (CTD) of EP400NL using bioinformatic and molecular modeling approaches and evaluated the anticancer potential of a peptide derived from this region.

Image Search Results

(A ) Schematic of the mitochondrial oxidative phosphorylation system, including the four enzymes comprising the electron transport chain (complexes I to IV) and the F1Fo-ATP synthase. The ADP/ATP translocator ANT and the phosphate carrier PiC are also depicted. The red lines indicate the site of action of specific inhibitors AA, antimycin A; ATR, atractyloside; BA, bongkrekic acid; Citreo, citreoviridin; KCN, potassium cyanide; Oligo, oligomycin; Rot, rotenone. ( B ) Effect of treatments for 80 min (50 min before the addition of 35S-methionine and through the 30 min pulse time) with the indicated OxPhos inhibitors on the incorporation of 35S-methionine into de novo synthesized mitochondrial polypeptides in whole HEK293T cells. ( C ) To follow the time-course effect of oligomycin on mitochondrial protein synthesis, cells were incubated with 2 μM oligomycin for 50 or 10 min before the addition of 35S-methionine and through the 30 min pulse time, or 10 min into the pulse and only through the last 20 min of labelling (see timeline scheme). In (B) and (C), cells were incubated in the presence of emetine to inhibit cytoplasmic protein synthesis. The proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to X-ray film, after which the signal was developed by autoradiography. Polypeptides synthesized by mitochondrial ribosomes are indicated on the left side. Immunoblotting against ACTIN was used as a loading control. The graphs represent the quantification of protein synthesis (PS) signal by densitometry, normalized to ACTIN, across three independent experiments. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3), One-way Anova with Dunnett multiple comparisons. ****: p<0.0001. ( D ) Time-course and dose-response assays on the inhibitory effect of oligomycin on ATPase activity. ( E-F ) Mitochondrial membrane potential (DYm) in HEK293T WT cells, treated or not with indicated OxPhos inhibitors. The assay measures the accumulation of tetramethylrhodamine methyl ester (TMRM) into mitochondria using flow cytometry. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 4-5)., One-way ANOVA with Dunnett multiple comparisons. ****: p<0.0001; ***: p<0.001, **: p<0.01, *: p<0.05.

Journal: bioRxiv

Article Title: Matrix nucleotide homeostasis couples energetic state to mitochondrial translation

doi: 10.64898/2026.06.04.730174

Figure Lengend Snippet: (A ) Schematic of the mitochondrial oxidative phosphorylation system, including the four enzymes comprising the electron transport chain (complexes I to IV) and the F1Fo-ATP synthase. The ADP/ATP translocator ANT and the phosphate carrier PiC are also depicted. The red lines indicate the site of action of specific inhibitors AA, antimycin A; ATR, atractyloside; BA, bongkrekic acid; Citreo, citreoviridin; KCN, potassium cyanide; Oligo, oligomycin; Rot, rotenone. ( B ) Effect of treatments for 80 min (50 min before the addition of 35S-methionine and through the 30 min pulse time) with the indicated OxPhos inhibitors on the incorporation of 35S-methionine into de novo synthesized mitochondrial polypeptides in whole HEK293T cells. ( C ) To follow the time-course effect of oligomycin on mitochondrial protein synthesis, cells were incubated with 2 μM oligomycin for 50 or 10 min before the addition of 35S-methionine and through the 30 min pulse time, or 10 min into the pulse and only through the last 20 min of labelling (see timeline scheme). In (B) and (C), cells were incubated in the presence of emetine to inhibit cytoplasmic protein synthesis. The proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to X-ray film, after which the signal was developed by autoradiography. Polypeptides synthesized by mitochondrial ribosomes are indicated on the left side. Immunoblotting against ACTIN was used as a loading control. The graphs represent the quantification of protein synthesis (PS) signal by densitometry, normalized to ACTIN, across three independent experiments. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3), One-way Anova with Dunnett multiple comparisons. ****: p<0.0001. ( D ) Time-course and dose-response assays on the inhibitory effect of oligomycin on ATPase activity. ( E-F ) Mitochondrial membrane potential (DYm) in HEK293T WT cells, treated or not with indicated OxPhos inhibitors. The assay measures the accumulation of tetramethylrhodamine methyl ester (TMRM) into mitochondria using flow cytometry. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 4-5)., One-way ANOVA with Dunnett multiple comparisons. ****: p<0.0001; ***: p<0.001, **: p<0.01, *: p<0.05.

Article Snippet: Human HEK293T embryonic kidney cells (CRL-3216), osteosarcoma 143B (CRL-8303), glioblastoma U-87 MG (HTB-14 ) , and neonatal fibroblasts (CCD-1064Sk) were obtained from the American Type Culture Collection (ATCC).

Techniques: Phospho-proteomics, Synthesized, Incubation, SDS Page, Membrane, Autoradiography, Western Blot, Control, Activity Assay, Flow Cytometry

( A ) Effect of the indicated OxPhos inhibitors on ATPase activity. ( B ) Mitochondrial membrane potential (ΔΨm) in HEK293T WT cells, treated or not with indicated OxPhos inhibitors. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3), One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. ( C and E ) Effect of treatments for 80 min (50 min before the addition of 35 S-methionine and through the 30 min pulse time) with the indicated compounds on the incorporation of 35 S-methionine into de novo synthesized mitochondrial polypeptides in whole HEK293T cells, in the presence of emetine to inhibit cytoplasmic protein synthesis. Polypeptides synthesized by mitochondrial ribosomes are indicated on the left side. Immunoblotting against ACTIN was used as a loading control. The graphs represent the quantification by densitometry of the protein synthesis (PS) signal normalized by ACTIN across three independent experiments. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3). One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, ****: p<0.0001. ( D ) Mitochondrial ATP levels assessed by the ATP bioluminescence assay (free Mg 2+ -ATP) or LC/MS (total ATP) in mitochondria isolated from HEK293T WT cells treated with the indicated OxPhos inhibitors. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3-9), One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

Journal: bioRxiv

Article Title: Matrix nucleotide homeostasis couples energetic state to mitochondrial translation

doi: 10.64898/2026.06.04.730174

Figure Lengend Snippet: ( A ) Effect of the indicated OxPhos inhibitors on ATPase activity. ( B ) Mitochondrial membrane potential (ΔΨm) in HEK293T WT cells, treated or not with indicated OxPhos inhibitors. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3), One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. ( C and E ) Effect of treatments for 80 min (50 min before the addition of 35 S-methionine and through the 30 min pulse time) with the indicated compounds on the incorporation of 35 S-methionine into de novo synthesized mitochondrial polypeptides in whole HEK293T cells, in the presence of emetine to inhibit cytoplasmic protein synthesis. Polypeptides synthesized by mitochondrial ribosomes are indicated on the left side. Immunoblotting against ACTIN was used as a loading control. The graphs represent the quantification by densitometry of the protein synthesis (PS) signal normalized by ACTIN across three independent experiments. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3). One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, ****: p<0.0001. ( D ) Mitochondrial ATP levels assessed by the ATP bioluminescence assay (free Mg 2+ -ATP) or LC/MS (total ATP) in mitochondria isolated from HEK293T WT cells treated with the indicated OxPhos inhibitors. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3-9), One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

Techniques: Activity Assay, Membrane, Synthesized, Western Blot, Control, ATP Bioluminescent Assay, Liquid Chromatography with Mass Spectroscopy, Isolation

( A-B ) Metagene plots of ribosome-protected fragments (RPFs) mapped to the mitochondrial genome in HEK293T cells treated with DMSO (upper panels, blue tracks) or oligomycin (lower panels, red tracks). Read densities are expressed as reads per million (RPM), scaled to codon positions. Gene positions are shown below the plots, with darker shading marking the first of overlapping open reading frames. ORFs encoded on the heavy (sense) and light (anti-sense) strands are indicated by the direction of the boxed arrows; the single light-strand ORF ( ND6 ) is shown in dark grey. (A) RPFs of 28-35 nt, and (B) RPFs of 20-24 nt are displayed. ( C ) Codon-specific ribosome occupancy profiles under DMSO and oligomycin conditions. Relative enrichment is plotted for each codon (color-coded by amino acid).

Journal: bioRxiv

Article Title: Matrix nucleotide homeostasis couples energetic state to mitochondrial translation

doi: 10.64898/2026.06.04.730174

Figure Lengend Snippet: ( A-B ) Metagene plots of ribosome-protected fragments (RPFs) mapped to the mitochondrial genome in HEK293T cells treated with DMSO (upper panels, blue tracks) or oligomycin (lower panels, red tracks). Read densities are expressed as reads per million (RPM), scaled to codon positions. Gene positions are shown below the plots, with darker shading marking the first of overlapping open reading frames. ORFs encoded on the heavy (sense) and light (anti-sense) strands are indicated by the direction of the boxed arrows; the single light-strand ORF ( ND6 ) is shown in dark grey. (A) RPFs of 28-35 nt, and (B) RPFs of 20-24 nt are displayed. ( C ) Codon-specific ribosome occupancy profiles under DMSO and oligomycin conditions. Relative enrichment is plotted for each codon (color-coded by amino acid).

Techniques:

( A ) Total mitochondrial GTP levels assessed by LC/MS in mitochondria isolated from HEK293T WT cells, expressing or not Saccharomyces cerevisiae GGC1, treated for 80 min with the indicated OxPhos inhibitors or pre-treated with ATR for 16h. This analysis was performed in parallel to the ATP measurements presented in . Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3-9), One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. ( B ) Schematic illustrating the strategy to selectively increase matrix GTP independently of adenine nucleotide exchange. HEK293T cells expressing empty vector, wild-type yeast mitochondrial GTP/GDP carrier (Ggc1), or an empty vector (EV) were treated with oligomycin (Oligo), oligomycin plus FCCP, or oligomycin + FCCP + bongkrekic acid (BA). FCCP was used to normalize membrane potential (ΔΨm), and BA to inhibit ANT-mediated adenine nucleotide exchange. Mitochondrial translation was assessed by 35 S-methionine incorporation in the presence of emetine. ( C ) Representative autoradiograph of 35 S-methionine incorporation into de novo synthesized mitochondrial polypeptides in HEK293T cells expressing vector or Ggc1, treated as indicated. Cytosolic translation was inhibited with emetine. ACTIN immunoblot serves as loading control. The graph shows the densitometric analysis of total 35 S-methionine incorporation normalized to ACTIN from n = X independent experiments. Data are mean ± SD. One-way ANOVA with Tukey (or Dunnett) multiple comparisons. **P < 0.01; ***P < 0.001; ****P < 0.0001. ( D ) Quantification by densitometry of the protein synthesis (PS) signal in panel (C), normalized by ACTIN across three independent experiments. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 2-3). One-way ANOVA with Dunnett multiple comparisons. ****: p<0.0001. ( E ) Model of mitochondrial translational control by matrix nucleotide phosphorylation potential. Inhibition of the F₁F₀-ATP synthase by oligomycin blocks proton re-entry into the mitochondrial matrix, causing membrane hyperpolarization (ΔΨ↑). The elevated ΔΨm impairs adenine nucleotide exchange across the inner mitochondrial membrane, limiting ATP import and retention in the matrix. Reduced matrix ATP compromises nucleoside diphosphate kinase (NDPK)-mediated regeneration of GTP from GDP, leading to depletion of the mitochondrial GTP pool. Because mitochondrial translation initiation and elongation require GTP-dependent factors (mtIF2, mtEF-Tu, mtEF-G), reduced GTP availability is expected to limit mitochondrial translation by restricting the activity of GTP-dependent translation factors.

Journal: bioRxiv

Article Title: Matrix nucleotide homeostasis couples energetic state to mitochondrial translation

doi: 10.64898/2026.06.04.730174

Figure Lengend Snippet: ( A ) Total mitochondrial GTP levels assessed by LC/MS in mitochondria isolated from HEK293T WT cells, expressing or not Saccharomyces cerevisiae GGC1, treated for 80 min with the indicated OxPhos inhibitors or pre-treated with ATR for 16h. This analysis was performed in parallel to the ATP measurements presented in . Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3-9), One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. ( B ) Schematic illustrating the strategy to selectively increase matrix GTP independently of adenine nucleotide exchange. HEK293T cells expressing empty vector, wild-type yeast mitochondrial GTP/GDP carrier (Ggc1), or an empty vector (EV) were treated with oligomycin (Oligo), oligomycin plus FCCP, or oligomycin + FCCP + bongkrekic acid (BA). FCCP was used to normalize membrane potential (ΔΨm), and BA to inhibit ANT-mediated adenine nucleotide exchange. Mitochondrial translation was assessed by 35 S-methionine incorporation in the presence of emetine. ( C ) Representative autoradiograph of 35 S-methionine incorporation into de novo synthesized mitochondrial polypeptides in HEK293T cells expressing vector or Ggc1, treated as indicated. Cytosolic translation was inhibited with emetine. ACTIN immunoblot serves as loading control. The graph shows the densitometric analysis of total 35 S-methionine incorporation normalized to ACTIN from n = X independent experiments. Data are mean ± SD. One-way ANOVA with Tukey (or Dunnett) multiple comparisons. **P < 0.01; ***P < 0.001; ****P < 0.0001. ( D ) Quantification by densitometry of the protein synthesis (PS) signal in panel (C), normalized by ACTIN across three independent experiments. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 2-3). One-way ANOVA with Dunnett multiple comparisons. ****: p<0.0001. ( E ) Model of mitochondrial translational control by matrix nucleotide phosphorylation potential. Inhibition of the F₁F₀-ATP synthase by oligomycin blocks proton re-entry into the mitochondrial matrix, causing membrane hyperpolarization (ΔΨ↑). The elevated ΔΨm impairs adenine nucleotide exchange across the inner mitochondrial membrane, limiting ATP import and retention in the matrix. Reduced matrix ATP compromises nucleoside diphosphate kinase (NDPK)-mediated regeneration of GTP from GDP, leading to depletion of the mitochondrial GTP pool. Because mitochondrial translation initiation and elongation require GTP-dependent factors (mtIF2, mtEF-Tu, mtEF-G), reduced GTP availability is expected to limit mitochondrial translation by restricting the activity of GTP-dependent translation factors.

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Expressing, Plasmid Preparation, Membrane, Autoradiography, Synthesized, Western Blot, Control, Phospho-proteomics, Inhibition, Activity Assay

USP11 directly interacts with GSK3β. (A) Volcano plot of proteins detected after USP11 immunoprecipitation from mouse mPFC. Log2 fold change (x-axis) shows enrichment versus control; log2 intensity (y-axis) reflects normalized quantitation in experimental samples. USP11 served as bait; GSK3β is highlighted as an interactor (log2 intensity USP11 = 22.9, log2FC = 2.38). (B) Immunoprecipitation (IP) with anti-USP11 antibody, immunoblot (IB) detection for USP11 (110 kDa) and GSK3β (47 kDa). IP with anti-GSK3β or anti-USP11 antibody. Input: whole lysate; IgG: isotype control. (C) Validation in HEK293T transfection system: lysates of vector control or Flag-USP11 transfected cells (Flag tag, 110 kDa) subjected to IP (anti-GSK3β), IB for anti-USP11. (D) Cell lysate analysis of HEK293T single His-GSK3β, single Flag-USP11, or co-transfected groups, immunoblotted for His-GSK3β (47 kDa) and Flag-USP11 (110 kDa). (E, F) Reciprocal Co-IP verification from HEK293T co-transfection. Immunoblot analysis for His and Flag tag in His-GSK3β, Flag-USP11, and co-transfected samples. (E) Lane 1: His-GSK3β group (IP-His), Lane 2: Flag-USP11 group (IP-Flag), Lane 3: Co-transfection (IP- His) (F) Lane 1: His -GSK3β group (IP- His), Lane 2: Flag-USP11 group (IP-Flag), Lane 3: Co-transfection (IP-Flag). (G) Dot blot analysis showing specific binding between USP11 and GSK3β. BSA (100/200/500 ng) served as negative control, and purified USP11 (100/200/500 ng) was spotted on the same nitrocellulose membrane. After incubation with GSK3β protein solution, binding was detected by fluorescence imaging. (H) Immunofluorescence analysis of co-localization: Exogenous expression in HEK293T cells demonstrates USP11 (red) and GSK3β (green); endogenous expression verified in primary neurons. Nuclei stained with DAPI (blue), scale bar = 25 μm. (I) Fluorescence intensity profiles along linear ROIs: Gray values of USP11 (red) and GSK3β (green) measured with ImageJ. Dual-channel curves plotted in GraphPad Prism using exported data. (J) Pearson's correlation scatter plots for USP11(red) and GSK3β(green) fluorescence, generated using ScatterJ plugin for ImageJ. Pearson's r value shown. (K) Schematic of Flag-tagged USP11 fragment constructs used for pulldown mapping. (L) HEK293T cells were co-transfected with Flag-USP11 or its deletion mutant and His- GSK3β, followed by immunoprecipitation and immunoblot analysis for Flag and His. (M) Computational molecular docking predicts multiple direct contact sites between USP11 and GSK3β.

Journal: Neurobiology of Stress

Article Title: USP11 drives stress-induced synaptic structural deficits and depression-like behaviors through GSK3β/mTOR signaling

doi: 10.1016/j.ynstr.2026.100791

Figure Lengend Snippet: USP11 directly interacts with GSK3β. (A) Volcano plot of proteins detected after USP11 immunoprecipitation from mouse mPFC. Log2 fold change (x-axis) shows enrichment versus control; log2 intensity (y-axis) reflects normalized quantitation in experimental samples. USP11 served as bait; GSK3β is highlighted as an interactor (log2 intensity USP11 = 22.9, log2FC = 2.38). (B) Immunoprecipitation (IP) with anti-USP11 antibody, immunoblot (IB) detection for USP11 (110 kDa) and GSK3β (47 kDa). IP with anti-GSK3β or anti-USP11 antibody. Input: whole lysate; IgG: isotype control. (C) Validation in HEK293T transfection system: lysates of vector control or Flag-USP11 transfected cells (Flag tag, 110 kDa) subjected to IP (anti-GSK3β), IB for anti-USP11. (D) Cell lysate analysis of HEK293T single His-GSK3β, single Flag-USP11, or co-transfected groups, immunoblotted for His-GSK3β (47 kDa) and Flag-USP11 (110 kDa). (E, F) Reciprocal Co-IP verification from HEK293T co-transfection. Immunoblot analysis for His and Flag tag in His-GSK3β, Flag-USP11, and co-transfected samples. (E) Lane 1: His-GSK3β group (IP-His), Lane 2: Flag-USP11 group (IP-Flag), Lane 3: Co-transfection (IP- His) (F) Lane 1: His -GSK3β group (IP- His), Lane 2: Flag-USP11 group (IP-Flag), Lane 3: Co-transfection (IP-Flag). (G) Dot blot analysis showing specific binding between USP11 and GSK3β. BSA (100/200/500 ng) served as negative control, and purified USP11 (100/200/500 ng) was spotted on the same nitrocellulose membrane. After incubation with GSK3β protein solution, binding was detected by fluorescence imaging. (H) Immunofluorescence analysis of co-localization: Exogenous expression in HEK293T cells demonstrates USP11 (red) and GSK3β (green); endogenous expression verified in primary neurons. Nuclei stained with DAPI (blue), scale bar = 25 μm. (I) Fluorescence intensity profiles along linear ROIs: Gray values of USP11 (red) and GSK3β (green) measured with ImageJ. Dual-channel curves plotted in GraphPad Prism using exported data. (J) Pearson's correlation scatter plots for USP11(red) and GSK3β(green) fluorescence, generated using ScatterJ plugin for ImageJ. Pearson's r value shown. (K) Schematic of Flag-tagged USP11 fragment constructs used for pulldown mapping. (L) HEK293T cells were co-transfected with Flag-USP11 or its deletion mutant and His- GSK3β, followed by immunoprecipitation and immunoblot analysis for Flag and His. (M) Computational molecular docking predicts multiple direct contact sites between USP11 and GSK3β.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Immunoprecipitation, Control, Quantitation Assay, Western Blot, Biomarker Discovery, Transfection, Plasmid Preparation, FLAG-tag, Co-Immunoprecipitation Assay, Cotransfection, Dot Blot, Binding Assay, Negative Control, Purification, Membrane, Incubation, Fluorescence, Imaging, Immunofluorescence, Expressing, Staining, Generated, Construct, Mutagenesis

USP11 regulates GSK3β ubiquitination, phosphorylation, and synaptic protein homeostasis in neural cells (A) Western blot analysis of GSK3β ubiquitination in HEK293T cells co-transfected with Flag-vector (control), Flag-USP11 (wild-type, 110 kDa), or Flag-USP11-C318S (catalytically inactive mutant). Endogenous GSK3β and phosphorylated GSK3β at Ser9 were immunoprecipitated from cell lysates using anti-GSK3β antibody, and ubiquitination levels were detected by immunoblotting with anti-ubiquitin antibody. GSK3β: 47 kDa; ubiquitin bands detected as smear. (B) Western blot analysis of GSK3β phosphorylation in three 293T cell groups: wild-type (Ctrl), stable USP11-overexpressing line generated by lentiviral transduction (USP11-OE), and USP11-overexpressing cells subjected to siRNA knockdown (USP11-OE + siUSP11). siUSP11 was transfected to silence USP11 in the stable overexpressing cell line. Whole cell lysates were analyzed for endogenous USP11 (110 kDa), phosphorylated GSK3β at Ser9 (p-GSK3β, 47 kDa), total GSK3β (47 kDa), and GAPDH (35 kDa) as loading control. Representative results from n = 3 biological replicates per group. (C) Gray value quantification of p-GSK3β/t-GSK3β in 293T cells (n = 3, F (2, 6) = 35.38, p = 0.0005). (D) Western blot analysis of USP11 (110 kDa), phosphorylated mTOR (p-mTOR, Ser2448, 289 kDa), total mTOR (289 kDa), p-GSK3β (Ser9, 47 kDa), total GSK3β (47 kDa), and Tubulin (55 kDa) in primary neurons upon USP11 siRNA knockdown (n = 3). (E, F) Gray value quantification of p-GSK3β/t-GSK3β, and p-mTOR/t-mTOR ratios in neurons upon USP11 siRNA knockdown (n = 3, p-GSK3β, p = 0.0213, p-mTOR, p = 0.0047). (G) Immunoblot of USP11 (110 kDa), p-GSK3β (Ser9, 47 kDa), total GSK3β (47 kDa), SYN (77 kDa), and Tubulin (55 kDa) in primary neurons infected with adeno-associated virus (AAV) (n = 3). (H, I) Gray value quantification of p-GSK3β/t-GSK3β, and SYN/Tubulin ratios in neurons transduced with vector or AAV-USP11 viruses (n = 3, p-GSK3β, p = 0.0078, SYN, Welch's t -test, p = 0.0031). (J) Representative immunofluorescence of primary neurons transduced with vector or AAV-USP11 viruses, showing DAPI (blue, nuclei), SYN (green, synaptophysin), and USP11 (magenta); merged panels display synapse integrity. Scale bar: 50 μm. (K, L) Quantitative analysis from three independent biological replicates in primary neurons transduced with vector or AAV-USP11 viruses (K) Mean USP11 immunofluorescence intensity (p = 0.0416), (L) Mean SYN immunofluorescence intensity (p = 0.0035). Data are shown as mean ± SEM. Determined by t -test (baseline comparisons) or one-way ANOVA (multiple groups) unless otherwise indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Journal: Neurobiology of Stress

Article Title: USP11 drives stress-induced synaptic structural deficits and depression-like behaviors through GSK3β/mTOR signaling

doi: 10.1016/j.ynstr.2026.100791

Figure Lengend Snippet: USP11 regulates GSK3β ubiquitination, phosphorylation, and synaptic protein homeostasis in neural cells (A) Western blot analysis of GSK3β ubiquitination in HEK293T cells co-transfected with Flag-vector (control), Flag-USP11 (wild-type, 110 kDa), or Flag-USP11-C318S (catalytically inactive mutant). Endogenous GSK3β and phosphorylated GSK3β at Ser9 were immunoprecipitated from cell lysates using anti-GSK3β antibody, and ubiquitination levels were detected by immunoblotting with anti-ubiquitin antibody. GSK3β: 47 kDa; ubiquitin bands detected as smear. (B) Western blot analysis of GSK3β phosphorylation in three 293T cell groups: wild-type (Ctrl), stable USP11-overexpressing line generated by lentiviral transduction (USP11-OE), and USP11-overexpressing cells subjected to siRNA knockdown (USP11-OE + siUSP11). siUSP11 was transfected to silence USP11 in the stable overexpressing cell line. Whole cell lysates were analyzed for endogenous USP11 (110 kDa), phosphorylated GSK3β at Ser9 (p-GSK3β, 47 kDa), total GSK3β (47 kDa), and GAPDH (35 kDa) as loading control. Representative results from n = 3 biological replicates per group. (C) Gray value quantification of p-GSK3β/t-GSK3β in 293T cells (n = 3, F (2, 6) = 35.38, p = 0.0005). (D) Western blot analysis of USP11 (110 kDa), phosphorylated mTOR (p-mTOR, Ser2448, 289 kDa), total mTOR (289 kDa), p-GSK3β (Ser9, 47 kDa), total GSK3β (47 kDa), and Tubulin (55 kDa) in primary neurons upon USP11 siRNA knockdown (n = 3). (E, F) Gray value quantification of p-GSK3β/t-GSK3β, and p-mTOR/t-mTOR ratios in neurons upon USP11 siRNA knockdown (n = 3, p-GSK3β, p = 0.0213, p-mTOR, p = 0.0047). (G) Immunoblot of USP11 (110 kDa), p-GSK3β (Ser9, 47 kDa), total GSK3β (47 kDa), SYN (77 kDa), and Tubulin (55 kDa) in primary neurons infected with adeno-associated virus (AAV) (n = 3). (H, I) Gray value quantification of p-GSK3β/t-GSK3β, and SYN/Tubulin ratios in neurons transduced with vector or AAV-USP11 viruses (n = 3, p-GSK3β, p = 0.0078, SYN, Welch's t -test, p = 0.0031). (J) Representative immunofluorescence of primary neurons transduced with vector or AAV-USP11 viruses, showing DAPI (blue, nuclei), SYN (green, synaptophysin), and USP11 (magenta); merged panels display synapse integrity. Scale bar: 50 μm. (K, L) Quantitative analysis from three independent biological replicates in primary neurons transduced with vector or AAV-USP11 viruses (K) Mean USP11 immunofluorescence intensity (p = 0.0416), (L) Mean SYN immunofluorescence intensity (p = 0.0035). Data are shown as mean ± SEM. Determined by t -test (baseline comparisons) or one-way ANOVA (multiple groups) unless otherwise indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Control, Mutagenesis, Immunoprecipitation, Generated, Transduction, Knockdown, Infection, Virus, Immunofluorescence

(A) Schematic of the experimental workflow: transcripts of varying architecture and length— including linear eGFP mRNA (∼1 kb), PB mRNA (2.1 kb), and circular eGFP RNA (circRNA, ∼1.7 kb) - were synthesized utilizing the specified RNAP mutants at 0, 100, or 150 mM NaCl. These preparations were transfected into HEK293T and THP-1 cell lines to evaluate translational efficiency and innate immune activation (via IFN-β secretion). (B, E) Mean fluorescence intensity (MFI) of eGFP expression in HEK293T cells at 48 and 96 h post-transfection for linear mRNA (B) and circRNA (E). (C, F) IFN-β secretory profiles of THP-1 cells following stimulation with linear mRNA (C) and circRNA (F) preparations. (D, G) Quantitative assessment of residual dsRNA content within linear mRNA (D) and circRNA (G) cohorts. (H) Longitudinal eGFP expression over 288 h; upper panel summarizes four circRNA mutants with distinct splicing introns, lower panels show individual trajectories. Notably, DBD-tethered chimeras maintained robust translational potency and negligible immunogenic signatures across all ionic strengths. Conversely, wild-type (WT) T7 and the T7(G47A/P884G) mutant exhibited a marked attenuation in transcriptional performance and product bioactivity under elevated salt conditions. Data are expressed as mean ±s.d. (n=3).

Journal: bioRxiv

Article Title: An Engineered Halotolerant Chimeric T7 RNA Polymerase for High-Yield, Low-Immunogenicity Synthesis of RNA via Simple Batch Transcription

doi: 10.64898/2026.05.13.724829

Figure Lengend Snippet: (A) Schematic of the experimental workflow: transcripts of varying architecture and length— including linear eGFP mRNA (∼1 kb), PB mRNA (2.1 kb), and circular eGFP RNA (circRNA, ∼1.7 kb) - were synthesized utilizing the specified RNAP mutants at 0, 100, or 150 mM NaCl. These preparations were transfected into HEK293T and THP-1 cell lines to evaluate translational efficiency and innate immune activation (via IFN-β secretion). (B, E) Mean fluorescence intensity (MFI) of eGFP expression in HEK293T cells at 48 and 96 h post-transfection for linear mRNA (B) and circRNA (E). (C, F) IFN-β secretory profiles of THP-1 cells following stimulation with linear mRNA (C) and circRNA (F) preparations. (D, G) Quantitative assessment of residual dsRNA content within linear mRNA (D) and circRNA (G) cohorts. (H) Longitudinal eGFP expression over 288 h; upper panel summarizes four circRNA mutants with distinct splicing introns, lower panels show individual trajectories. Notably, DBD-tethered chimeras maintained robust translational potency and negligible immunogenic signatures across all ionic strengths. Conversely, wild-type (WT) T7 and the T7(G47A/P884G) mutant exhibited a marked attenuation in transcriptional performance and product bioactivity under elevated salt conditions. Data are expressed as mean ±s.d. (n=3).

Article Snippet: HEK293T (Human Embryonic Kidney 293T cells) cells were obtained from ATCC and grown in Dulbecco’s Modified Eagle Medium (DMEM; Corning, USA) supplemented with 10% FBS (Gibco, USA) and 1% Penicillin-Streptomycin (P/S; ThemoFisher, USA).

Techniques: Synthesized, Transfection, Activation Assay, Fluorescence, Expressing, Mutagenesis

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

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

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.

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

$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.$

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

$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.$

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

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.

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

$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.$

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

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