wt construct  (Addgene inc)

Journal: PLOS One

Article Title: Characterization of p53 p.T253I as a pathogenic mutation underlying Li-Fraumeni Syndrome

doi: 10.1371/journal.pone.0320036

Figure Lengend Snippet: (A) HEK293 p53 -/- cells were stably complemented either WT-GFP, C176Y-GFP, R213X-GFP or T253I-GFP p53. After selection, cells were assayed for GFP fluorescence by flow cytometry. Shown are representative histograms (green fluorescence) of the geometric means of each complemented cell type. (B) Graphical representation of fluorescent intensity of each p53 cell line. Median GFP fluorescent intensity was calculated for each cell line and normalized to the p53 WT expression levels. Data represent the mean of three experimental replicates. (C) Cell growth kinetics of stably complemented HEK293 p53 -/- cells were measured by use of a CellTrace Violet assay. After incorporating a fluorescent dye, cells were harvested 24, 48, 72, and 96 hours after treatment, fixed, and dye intensity was measured by flow cytometry. Geometric mean intensity at each timepoint was used to calculate an exponential decay regression curve and ultimately a population doubling time (PDT). Data represent the mean PDT of three experimental replicates and significance is denoted by * or † (* indicates p < 0.05 when comparing the sample to p53 -/-, † p < 0.05 when comparing the sample to p53 WT).

Article Snippet: HEK TP53 wt/wt (ATCC Cat. #CRL-1573), HEK Crispr-deleted TP53 -null ( TP53 -/- ; Ubigene Cat. #YKO-H177), and all stably transduced derivatives of HEK TP53 -/- cells were maintained in DMEM (Thermo Scientific) supplemented with 10% FBS (Gemini Bio-Products) in a 37°C incubator with 5% CO 2 and maintained in log growth phase.

Techniques: Stable Transfection, Selection, Fluorescence, Flow Cytometry, Expressing

Journal: PLOS One

Article Title: Characterization of p53 p.T253I as a pathogenic mutation underlying Li-Fraumeni Syndrome

doi: 10.1371/journal.pone.0320036

Figure Lengend Snippet: HEK293 p53 -/- cells and those complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53 were subjected to subcellular fractionation to look at the distribution of p53 and MDM2 in the cytoplasm ( A, left side ) and nucleus ( A, right side ). (B) Graphical depictions of the cytoplasmic ( left ) and nuclear ( right ) expression levels of p53. (C) Graphical depictions of the cytoplasmic ( left ) and nuclear ( right ) expression levels of MDM2. In both cases, expression levels of cytoplasmic fractions were normalized to GAPDH while expression levels of nuclear fractions were normalized to Histone 3 (H3). Data represent the mean expression levels of three experimental replicates and significance is denoted by * or † (* indicates p < 0.05 when comparing the sample to p53 -/-, † p < 0.05 when comparing the sample to p53 WT).

Article Snippet: HEK TP53 wt/wt (ATCC Cat. #CRL-1573), HEK Crispr-deleted TP53 -null ( TP53 -/- ; Ubigene Cat. #YKO-H177), and all stably transduced derivatives of HEK TP53 -/- cells were maintained in DMEM (Thermo Scientific) supplemented with 10% FBS (Gemini Bio-Products) in a 37°C incubator with 5% CO 2 and maintained in log growth phase.

Techniques: Fractionation, Expressing

Journal: PLOS One

Article Title: Characterization of p53 p.T253I as a pathogenic mutation underlying Li-Fraumeni Syndrome

doi: 10.1371/journal.pone.0320036

Figure Lengend Snippet: HEK293 p53 -/- cells and those complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53 were treated with 5 ug/mL Cisplatin (or a DMSO vehicle control) for one hour followed by a 2-hour recovery period. Cells were collected and a Western blot was run looking at p53 signaling events. Levels of p53, MDM2, ( A, left side ) and p21 ( A, right side ) were examined with or without damage in each of the p53 conditions (null, WT, and the three mutants). Graphical depictions of p53 (B) , p21 (C) , and MDM2 ( D ) are shown. Data represent the mean expression levels, normalized to GAPDH, of three experimental replicates and significance is denoted by * or † (* indicates p < 0.05 when comparing the sample to p53 -/-, † p < 0.05 when comparing the sample to p53 WT).

Article Snippet: HEK TP53 wt/wt (ATCC Cat. #CRL-1573), HEK Crispr-deleted TP53 -null ( TP53 -/- ; Ubigene Cat. #YKO-H177), and all stably transduced derivatives of HEK TP53 -/- cells were maintained in DMEM (Thermo Scientific) supplemented with 10% FBS (Gemini Bio-Products) in a 37°C incubator with 5% CO 2 and maintained in log growth phase.

Techniques: Control, Western Blot, Expressing

Journal: PLOS One

Article Title: Characterization of p53 p.T253I as a pathogenic mutation underlying Li-Fraumeni Syndrome

doi: 10.1371/journal.pone.0320036

Figure Lengend Snippet: HEK293 p53 -/- cells and those complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53 were treated with 5 ug/mL Cisplatin (or a DMSO vehicle control) for one hour followed by a 2-hour recovery period. RNA was isolated from these cells and subjected to rtPCR in the presence of primers for p21 ( A ) and MDM2 (B) . Data represent the mean RNA expression levels of three experimental replicates and significance is denoted by * or † (* indicates p < 0.05 when comparing the sample to p53 -/-, † p < 0.05 when comparing the sample to p53 WT).

Article Snippet: HEK TP53 wt/wt (ATCC Cat. #CRL-1573), HEK Crispr-deleted TP53 -null ( TP53 -/- ; Ubigene Cat. #YKO-H177), and all stably transduced derivatives of HEK TP53 -/- cells were maintained in DMEM (Thermo Scientific) supplemented with 10% FBS (Gemini Bio-Products) in a 37°C incubator with 5% CO 2 and maintained in log growth phase.

Techniques: Control, Isolation, Reverse Transcription Polymerase Chain Reaction, RNA Expression

Journal: PLOS One

Article Title: Characterization of p53 p.T253I as a pathogenic mutation underlying Li-Fraumeni Syndrome

doi: 10.1371/journal.pone.0320036

Figure Lengend Snippet: HEK293 p53 -/- cells and those complemented with WT-GFP, C176Y-GFP, R213X-GFP, or T253I-GFP p53 were assayed for DNA binding capability and transcriptional regulatory capacity. (A) Log phase cells were exposed 5 µg/mL Cisplatin (or a DMSO vehicle control) for 24 hours. Nuclear protein was isolated from cells and a DNA binding assay was performed. Binding efficiency was measured by the ability of nuclear p53 to recognize and bind to consensus p53 binding DNA sequences attached to 96-well plate wells. Data represent the binding strength of each condition relative to undamaged p53 WT cells and are expressed as the mean of three experimental replicates. (B) DNA binding capacity of HEK p53 -/- and p53 -/- + WT, C176Y, R231X, or T253I p53 log phase cells was measured using CHIP-PCR. Genomic DNA was isolated, fragmented, and immunprecipitated with p53 antibody. After separating the DNA, primers to p21 (and RPL30 as a control) were used to identify the relative quantity of bound p21 promoter DNA in each p53 condition through PCR. Data represent the mean of three experimental replicates. (C) Transcriptional regulatory capacity was measured using a dual-glow luciferase assay. Cells were co-transfected with luc2P/p53 RE and hRluc/CMV vectors at a 10:1 ratio. After 24 hours the expression of these genes was measured by a stop-and-glow (Promega) system. For each p53 condition, luminescent intensity was calculated as a ratio of luc2P to hRluc and expressed as a percentage of intensity relative to WT-GFP-complemented p53 -/- cells. Data are expressed as the mean of three experimental replicates and significance in (A) ( B ) and ( C ) is denoted by * or † (* indicates p < 0.05 when comparing the sample to p53 -/-, † p < 0.05 when comparing the sample to p53 WT).

Article Snippet: HEK TP53 wt/wt (ATCC Cat. #CRL-1573), HEK Crispr-deleted TP53 -null ( TP53 -/- ; Ubigene Cat. #YKO-H177), and all stably transduced derivatives of HEK TP53 -/- cells were maintained in DMEM (Thermo Scientific) supplemented with 10% FBS (Gemini Bio-Products) in a 37°C incubator with 5% CO 2 and maintained in log growth phase.

Techniques: Binding Assay, Control, Isolation, DNA Binding Assay, Luciferase, Transfection, Expressing