hek 293t cells  (ATCC)

Journal: Nucleic Acids Research

Article Title: Mistranslation from an endogenous tRNA variant in human pan-genome cell lines

doi: 10.1093/nar/gkag224

Figure Lengend Snippet: Cytotoxicity and cell viability in mistranslating human and murine cells. Plasmids encoding mCherry and wild-type human tRNA Ser AGA or variant tRNA Ser AAA with different flanking sequence contexts from human tRNA Ser AGA 2–2, 2–3, 2–4, and 2–5 genes were transfected into ( A ) human HEK 293T and ( B ) murine N2a cells; ( A, B ) cytotoxicity was determined at 24 h post-transfection using CytotoxGlo. ( C, D ) Cell viability (CCK8 assay) was also measured in ( C ) HEK 293T cells and in ( D ) N2a cells at 24 h after transfection with a plasmid expressing the wildtype GFP-mCherry reporter and the indicated wildtype tRNA Ser AGA or AAA anticodon mutant. Error bars represent ±1 standard deviation of the mean and statistical analysis was calculated using single factor ANOVA and was based on at least N = 3 biological replicates (n.s., not significant; * P <.05).

Article Snippet: Mammalian cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States), and cell culture work was conducted in the human embryonic kidney (HEK) 293T cells (CRL-3216, ATCC), in murine neuroblastoma Neuro-2a (N2a) cells (CCL-131, ATCC), or in human K562 lymphoblast cells (CCL-243, ATCC) as indicated.

Techniques: Variant Assay, Sequencing, Transfection, CCK-8 Assay, Plasmid Preparation, Expressing, Mutagenesis, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Mistranslation from an endogenous tRNA variant in human pan-genome cell lines

doi: 10.1093/nar/gkag224

Figure Lengend Snippet: Quantifying mistranslation levels in live human cells. HEK 293T cells were transfected with a plasmid co-expressing GFP fused to mCherry (WT) or to mCherry S151F and wildtype tRNA Ser AGA or mutant tRNA Ser AAA . ( A ) Representative images of brightfield, GFP, and mCherry fluorescence are shown alongside ( B ) zoomed-in views (white boxes in A) showing GFP, mCherry, and merged fluorescent images of the indicated cell lines. Western blotting of the reporter with mCherry and GAPDH (loading control) specific antibodies from cells expressing wildtype or anticodon variants of ( C ) tRNA Ser AGA 2–5 or ( D ) tRNA Ser AGA 2–3 show the full length GFP-mCherry protein (54 kDa) and a lower molecular weight proteolytic product that occurs in mCherry . ( E, F ) The mistranslation level is determined by plotting the ratio of mCherry/GFP fluorescence per cell. The images in ( A ) and in were quantified and normalized to the mCherry/GFP fluorescence ratio observed in mistranslated cells expressing the wildtype reporter. Error bars represent ±1 standard deviation of the mean and statistical analysis was calculated using single factor ANOVA and was based on N = 3 biological replicates (n.s., not significant; * P <.05; ** P <.01).

Article Snippet: Mammalian cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States), and cell culture work was conducted in the human embryonic kidney (HEK) 293T cells (CRL-3216, ATCC), in murine neuroblastoma Neuro-2a (N2a) cells (CCL-131, ATCC), or in human K562 lymphoblast cells (CCL-243, ATCC) as indicated.

Techniques: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Fluorescence, Western Blot, Control, Molecular Weight, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Mistranslation from an endogenous tRNA variant in human pan-genome cell lines

doi: 10.1093/nar/gkag224

Figure Lengend Snippet: Quantifying mistranslation levels in live human and murine cells by flow cytometry. Flow-cytometry was used to measure GFP and mCherry fluorescence in 2.5 × 10 6 individual viable HEK 293T (above) or murine N2a cells (below) transfected with the wildtype GFP-mCherry or GFP-mCherry S151F mutant and the indicated wildtype tRNA Ser AGA or tRNA Ser AAA variant. ( A ) The fluorescence measurements were converted into heatmaps to depict the population of cells observed across mCherry and GFP fluorescence levels. ( B ) To determine the mistranslation level resulting from each tRNA mutant in the indicated cell lines, the mCherry/GFP fluorescence ratio from individual cells were calculated and plotted. The data were normalized to the wildtype mCherry/GFP fluorescence ratio observed in mistranslating cells. Error bars represent ±1 standard deviation of the mean and statistical analysis was calculated using single factor ANOVA and was based on N = 3 biological replicates (n.s., not significant; ** P <.01; *** P <.001; **** P <.0001).

Article Snippet: Mammalian cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States), and cell culture work was conducted in the human embryonic kidney (HEK) 293T cells (CRL-3216, ATCC), in murine neuroblastoma Neuro-2a (N2a) cells (CCL-131, ATCC), or in human K562 lymphoblast cells (CCL-243, ATCC) as indicated.

Techniques: Flow Cytometry, Fluorescence, Transfection, Mutagenesis, Variant Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Mistranslation from an endogenous tRNA variant in human pan-genome cell lines

doi: 10.1093/nar/gkag224

Figure Lengend Snippet: Mistranslation levels in individual human cells as a function of dual reporter expression. Based on the data from the flow cytometry analysis (Fig. ), the logarithm of the mistranslation level observed in individual HEK 293T cells expressing the tRNA Ser AAA ( A ) 2–3 or ( B ) 2–5 variant was plotted as a function of the GFP fluorescence level observed in the same cells. ( C ) The plots were overlaid to highlight reduced GFP fluorescence levels in the cells expressing the 2–5 variant (as in ) and reveal a nearly identical inverse power-law relationship (tRNA Ser AAA 2–3 blue solid line; tRNA Ser AAA 2–5 dark orange dashed line) between mistranslation level and the expression level of the reporter for both tRNA variants.

Article Snippet: Mammalian cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States), and cell culture work was conducted in the human embryonic kidney (HEK) 293T cells (CRL-3216, ATCC), in murine neuroblastoma Neuro-2a (N2a) cells (CCL-131, ATCC), or in human K562 lymphoblast cells (CCL-243, ATCC) as indicated.

Techniques: Expressing, Flow Cytometry, Variant Assay, Fluorescence

Journal: Nucleic Acids Research

Article Title: Mistranslation from an endogenous tRNA variant in human pan-genome cell lines

doi: 10.1093/nar/gkag224

Figure Lengend Snippet: Mass spectrometry of GFP-mCherry S151F isolated from human cells. The mCherry S151F protein was isolated from HEK 293T cells expressing the indicated wildtype tRNA Ser AGA or G35A anticodon variant. Y and b ion spectra of mistranslated peptides (above, f = Ser incorporated at Phe151) are compared to matching properly translated peptides (below, F = Phe incorporated at Phe151) derived from cells expressing ( A ) tRNA Ser AAA 2–3 or ( B ) tRNA Ser AAA 2–5. ( C ) Peptide coverage maps are shown for the peptide surrounding the Phe151 mutation site. Blue lines indicate the expected peptide sequence listed at the top, while the boxed f annotates Ser mis-incorporation at F151. The apparent level of Ser mis-incorporation at ( D ) Phe151 and ( E ) at all Phe codons in the GFP-mCherry protein was estimated from the mass spectrometry data based on the relative area under the monoisotopic peak for a mistranslated relative to a properly translated peptide found in the protein samples extracted from the indicated HEK 293T cell lines. ( F ) We also used spectral counts, i.e. the number of mistranslated relative to properly translated peptides identified in each sample, as an alternate approach to estimate mis-incorporation level at the Phe151 codon. Error bars represent ±1 standard deviation of the mean and statistical analysis was calculated using single factor ANOVA and was based on N = 3 biological replicates (n.s., not significant; * P <.05).

Article Snippet: Mammalian cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States), and cell culture work was conducted in the human embryonic kidney (HEK) 293T cells (CRL-3216, ATCC), in murine neuroblastoma Neuro-2a (N2a) cells (CCL-131, ATCC), or in human K562 lymphoblast cells (CCL-243, ATCC) as indicated.

Techniques: Mass Spectrometry, Isolation, Expressing, Variant Assay, Derivative Assay, Mutagenesis, Sequencing, Standard Deviation