human embryonic kidney 293t hek293t cells  (ATCC)

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

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.

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

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).

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:

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.

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

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

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

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: Identification of the ubiquitination site on RIG-I targeted by FGF8. (a) diagram illustrating the truncated constructs of RIG-I. (b) HEK-293T cells were co-transfected with specified plasmids and exposed to MG132 for 6 hours. Western blot analysis was conducted to assess the ubiquitination of various RIG-I truncation constructs. (C) Western blot analysis identified the ubiquitination site on RIG-I targeted by FGF8, and band intensities were quantified by densitometry to assess the degradation of each mutant. (d) a dual-luciferase assay was conducted in HEK293T cells co-transfected with specified RIG-I mutants and FGF8 to evaluate the impact of FGF8 on IFN-β promoter activity. Error bars indicate the mean ± SEM from three independent experiments. Two-tailed unpaired Student’s t-tests were used. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Human lung adenocarcinoma cell line A549 (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0016), human embryonic kidney cell line HEK293T (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0005), and Madin-Darby canine kidney cell line MDCK (Procell Life Science & Technology Co., Ltd., Wuhan, China; Cat. No. CL-0154) were used for virus infection experiments, protein interaction validation experiments, and virus titration assays, respectively.

Techniques: Ubiquitin Proteomics, Construct, Transfection, Western Blot, Mutagenesis, Luciferase, Activity Assay, Two Tailed Test

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