cell lines human embryonic kidney hek 293t cells  (ATCC)

Journal: Cell Chemical Biology

Article Title: Ligand-induced assembly of antibody variable fragments for the chemical regulation of biological processes

doi: 10.1016/j.chembiol.2025.01.007

Figure Lengend Snippet: Application of Fv-based CIDs for inducible regulation of gene expression (A) Scheme of an inducible CRISPRa-based system for regulation of gene expression. The transcriptional activator domain VPR and the catalytically inactive Cas9 protein (dCas9) are fused Fv variable domains (V L or V H ). Upon ligand addition, the VPR and dCas9 domains are brought together and activate the transcription of the firefly luciferase gene (fLuc) encoded on a reporter plasmid with 10 binding sites for gRNA upstream of the minimal promoter (pMIN). (B) Inducible activation of luciferase reporter gene using FvNIC (left) and FvEST (right) systems in HEK293T cells stimulated with 1 mM nicotine and 10 μM estradiol, respectively. (C and D) Construction and validation of OR gate (C) and AND gate (D) logic circuits using FvNIC and FvEST systems in combination with dCas9 DNA-binding domain. Cells were stimulated with 10 μM estradiol and/or 1 mM nicotine as indicated. Results in (B–D) are shown as fold activation in reporter gene expression compared to vehicle control mock cells transfected with reporter plasmid only. Plots in (B–D) show the means ± SD of n = 8 biological replicates combined from two independent experiments. (E) Activation of endogenous genes by FvNIC (cyan) and FvEST (orange) systems. Cells were transfected with individual gRNAs targeting TTN, ASCL1, and NEUROD1 genes and stimulated with indicated ligands. Cells transfected only with constitutively expressed dCas9:VPR fusion and indicated gRNAs served as a positive control to determine the maximum expected level of gene activation (dark gray bars). Results are shown as fold activation in reporter gene expression compared to vehicle control mock cells transfected with an empty plasmid (pcDNA3.1). Plots in (E) show the means ± SD of n = 6 biological replicates combined from two independent experiments. Conditions were compared using a two-sided unpaired t test with Welch correction (∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; ns > 0.05).

Article Snippet: The cell lines Human embryonic kidney (HEK) 293T cells (ATCC, HEK293T) and Lenti-X™293T cells (Takara-Bio, LentiX293) were cultured in DMEM medium (Thermo Fisher Scientific) supplemented with 10 % v/v FBS (Thermo Fisher Scientific).

Techniques: Gene Expression, Luciferase, Plasmid Preparation, Binding Assay, Activation Assay, Control, Transfection, Positive Control

Journal: Cell Chemical Biology

Article Title: Ligand-induced assembly of antibody variable fragments for the chemical regulation of biological processes

doi: 10.1016/j.chembiol.2025.01.007

Figure Lengend Snippet:

Article Snippet: The cell lines Human embryonic kidney (HEK) 293T cells (ATCC, HEK293T) and Lenti-X™293T cells (Takara-Bio, LentiX293) were cultured in DMEM medium (Thermo Fisher Scientific) supplemented with 10 % v/v FBS (Thermo Fisher Scientific).

Techniques: Virus, Recombinant, Expressing, Lysis, Membrane, Affinity Chromatography, Luciferase, Cell Isolation, Enzyme-linked Immunosorbent Assay, Isolation, SYBR Green Assay, Software

Journal: Scientific Reports

Article Title: Anti-tumor efficacy and safety of AEV01 in preclinical glioblastoma and hepatocellular carcinoma models

doi: 10.1038/s41598-025-89594-w

Figure Lengend Snippet: ( A – C ) Cellular viability of malignant GB, liver cancer and HEK-293T cells following AEV01 treatment for 24 h. Cell viability was assessed using MTT assay, n = 3.

Article Snippet: HEK-293T Human embryonic kidney cell line (RRID: CVCL_0063) (Passage No: 10), U-87 MG human GB cell line (RRID: CVCL_5J15) (Passage Number: 58), HepG2 human liver cancer cell line (RRID: CVCL_0027) (Passage Number: 35), Hep3B human liver cancer cell line (RRID: CVCL_0326) (Passage Number: 24) and WRL-68 liver senescence cells (RRID: CVCL_0581) (Passage Number: 68) were procured from National Centre for Cell Sciences (NCCS) (Pune, India).

Techniques: MTT Assay

Journal: Communications Biology

Article Title: Inhibition of SCF KDM2A /USP22-dependent nuclear β-catenin ubiquitylation mediates cerebral ischemic tolerance

doi: 10.1038/s42003-025-07644-5

Figure Lengend Snippet: A Western blot assay showing the expressions of total, nuclear and cytoplasmic β-catenin at 2, 6, 12, 24 h after OGD/R in in primary hippocampal neurons, respectively. GAPDH is used as loading control to normalize the relative expression of whole cellular/cytoplasmic β-catenin and Lamin B1 is used as loading control to normalize the relative expression of nuclear β-catenin. Data are expressed as percentage of value of the control group. * p < 0.05 versus control group ( p = 0.232 for total β-catenin, n = 3-5, Kruskal-Wallis Test; p < 0.001 for nuclear β-catenin, n = 6, One-Way ANOVA Test, p = 0.048 at OGD/R 2 h, p = 0.001 at OGD/R 6 h, p = 0.011 at OGD/R 12 h, p = 0.019 at OGD/R 24 h, Tamhane post-test; p = 0.345 for cytoplasmic β-catenin, n = 4, One-Way ANOVA Test). B Effects of MG132 on nuclear and cytoplasmic β-catenin proteins in primary hippocampal neurons at 24 h after OGD/R using western blot analysis. GAPDH and Lamin B1 are used as loading control to normalize the relative expression of cytoplasmic and nuclear β-catenin, respectively. Data are expressed as percentage of value of the control group with DMSO. * p < 0.05 versus the control (ctrl) group with DMSO ( p = 0.04 for nuclear β-catenin, p = 0.977 for cytoplasmic β-catenin, unpaired t -test), # p < 0.05 versus the same group with DMSO ( p < 0.001for nuclear β-catenin in the ctrl grou p or OGD/R group, p = 0.211 for cytoplasmic β-catenin in the ctrl group, p = 0.03 for cytoplasmic β-catenin at the OGD/R group, un p aired t-test, n = 3 in each group). C Immunoprecipitation assay showing the K48-Ub of nuclear β-catenin. β-catenin was immunoprecipitated (IP) using anti-β-catenin antibody. IgG antibody was used as a negative control. K48-Ub was detected by western blot using anti-ubiquitin (linkage-specific K48) antibody. Data are expressed as percentage of value of the control group with MG132. * p < 0.05 versus the control group with MG132 ( p = 0.018, n = 3-4, unpaired t -test). D Immunofluorescence assay showing that the effects of Flag-KDM2A (a-d, e-h) or Flag-KDM2A-∆F-box (i-l, m-p) plasmids treatment on the fluorescence intensity of β-catenin with or without MG132 treatment. Representative photomicrographs with fluorescent staining of β-catenin (white), Flag (red) and DAPI (blue) in SH-SY5Y cells. White arrows indicate the positive cells expressed exogenous KDM2A protein. Scale bar: 10 μm. E Western blot assay showing the expression of Flag, KDM2A and β-catenin in nuclear lysates of HEK-293T and SH-SY5Y cells which were transfected with the indicated plasmids and then treated with 20 μM MG132 or DMSO for 6 h. The histogram presents the quantitative analyses of nuclear β-catenin levels. PCNA is used as a loading control to normalize the relative expression of nuclear β-catenin. Data are expressed as percentage of value of control group with DMSO. * p < 0.05 versus Flag-Con group with DMSO ( p = 0.026 for HEK-293T, p = 0.026 in the Flag-KDM2A group with DMSO, p = 0.207 in the Flag-KDM2A-∆F-box group with DMSO; p = 0.008 for SH-SY5Y, p = 0.01 in the Flag-KDM2A group with DMSO, p = 0.05 at the Flag-KDM2A-∆F-box grou p with DMSO, One-Way ANOVA Test followed by Bonferroni post-test), # p < 0.05 versus the same group with DMSO (for HEK293T, p = 0.003 in the Flag-Con group, p = 0.005 in the Flag-KDM2A group, p < 0.001 in the Flag-KDM2A-∆F-box group; for SH-SY5Y, p = 0.036 in the Flag-Con group, p = 0.010 in the Flag-KDM2A group, p = 0.002 in the Flag-KDM2A-∆F-box group, unpaired t- test, n = 8 in each group). F Effects of Flag-KDM2A or Flag-KDM2A-∆F-box plasmids treatment on the ubiquitination of Myc-β-catenin were assessed by in vivo ubiquitination assay. HA-Ub and Myc-β-catenin were co-transfected into HEK-293T cells with KDM2A constructs or empty vector control. G Western blots and Immunoprecipitation blots showing the expression of SKP1, CUL1 and Flag and the assembly of SCF KDM2A ligase complex in nuclear lysates of HEK-293T cells which were transfected with the indicated plasmids. HNs, hippocampal neurons; Flag, flag-tagged protein. The nuclear fraction purity was assessed by absence of β-tubulin staining in Figure E– G . Each bar represents the mean ± SD (error bars).

Article Snippet: For cell cultures, human neuroblastoma SH-SY5Y cells and human embryonic kidney HEK-293T cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Control, Expressing, Immunoprecipitation, Negative Control, Immunofluorescence, Fluorescence, Staining, Transfection, In Vivo, Ubiquitin Assay, Construct, Plasmid Preparation