pg13 luc  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: An engineered glutamic acid tRNA for efficient suppression of pathogenic nonsense mutations

doi: 10.1093/nar/gkaf532

Figure Lengend Snippet: tRNA GluV13 rescues endogenous nonsense mutations in TP53 . ( A ) Representative western blots of cells transfected with an empty vector or a plasmid containing three copies of tRNA Pyl (control tRNA) or tRNA GluV13 CUA . Lysates were probed for p53 and CBP80 (loading control) 48 h after transfection. Bands corresponding to full-length (FL p53) and truncated (TR p53) are indicated. ( B ) p53-dependent luciferase activity in cells transfected with an empty vector or a plasmid containing three copies of tRNA Pyl (control tRNA) or tRNA GluV13 CUA . Data are displayed as the fold change in luciferase activity relative to cells transfected with an empty vector and reported as mean ± SD, n = 3. * P < 0.05, *** P < 0.0005, ns = not significant, unpaired Student’s t -test.

Article Snippet: The following day, cells were transfected with 50 ng of the indicated sup-tRNA plasmid (or an empty vector), 50 ng of p53-dependent Fluc reporter plasmid PG13-luc (a gift from Bert Vogelstein; Addgene Plasmid # 16442; http://n2t.net/addgene:16442; RRID:ADDGENE_16442 ), and 15 ng of pLX313- Renilla .

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay

Journal: International Journal of Cancer

Article Title: Transcriptional repression of SOX2 by p53 in cancer cells regulates cell identity and migration

doi: 10.1002/ijc.35490

Figure Lengend Snippet: Effects of altered TP53 expression on SOX2 levels. (A) TP53 (left), CDKN1A (middle) and SOX2 (right) relative mRNA levels by RT‐qPCR in TP53 defective NCI‐H1299, U‐373MG and T‐47D tumor cell lines overexpressing TP53 . (B) TP53, P21 and SRY‐box transcription factor 2 (SOX2) protein levels by western blot in TP53 defective NCI‐H1299, U‐373MG and T‐47D tumor cell lines overexpressing TP53 . (C) TP53 (left), CDKN1A (middle) and SOX2 (right) relative mRNA levels by RT‐qPCR in TP53 wild type A‐549, U‐87MG and MCF‐7 tumor cell lines with silenced TP53 expression. (D) TP53, CDKN1A and SOX2 protein levels by western blot in TP53 wild type A‐549, U‐87MG and MCF‐7 tumor cell lines with silenced TP53 expression. P ‐values less than 0.05 were considered statistically significant: *** p < .001; ** p < .01; * p < .05; ns not significant.

Article Snippet: To analyze the activity of regulatory regions, cells were co‐transfected with luciferase reporter plasmids PG13‐luc (wt p53 binding sites) (a gift from Bert Vogelstein; Addgene #16442) and pGL3‐Sox2 luc (a gift from Richard G. Pestell) in the presence of PEI.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: International Journal of Cancer

Article Title: Transcriptional repression of SOX2 by p53 in cancer cells regulates cell identity and migration

doi: 10.1002/ijc.35490

Figure Lengend Snippet: The alteration of TP53 levels modulates SOX2 activity transcriptionally in lung cancer and glioma cell lines. (A) Luciferase activity from the CDKN1A promoter (pG13‐luc) or the SOX2 promoter (pGl3‐Sox2‐luc) in TP53 defective tumor cell lines NCI‐H1299 and U‐373MG overexpressing TP53 (top) and in TP53 wild type cell lines A‐549 and U87MG (bottom). (B) Analysis of TP53 binding by Trp53 induction with 0.5 μg/mL doxorubicin for 10 h followed by chromatin immunoprecipitation with a Trp53 antibody and quantitative real‐time PCR of the Cdkn1a promoter, the Sox2 promoter, the Sox2 Regulatory Region 1 ( SRR1 ) and the Sox2 Regulatory Region 2 ( SRR2 ) (with two different primer pairs). EV, empty vector. P ‐values less than 0.05 were considered statistically significant: *** p < .001; ** p < .01; * p < .05; ns not significant.

Article Snippet: To analyze the activity of regulatory regions, cells were co‐transfected with luciferase reporter plasmids PG13‐luc (wt p53 binding sites) (a gift from Bert Vogelstein; Addgene #16442) and pGL3‐Sox2 luc (a gift from Richard G. Pestell) in the presence of PEI.

Techniques: Activity Assay, Luciferase, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: International Journal of Cancer

Article Title: Transcriptional repression of SOX2 by p53 in cancer cells regulates cell identity and migration

doi: 10.1002/ijc.35490

Figure Lengend Snippet: Increasing SOX2 levels promotes cell migration capacity. Representative images and quantification of the transwell migration assay of A‐549 (top) and MCF‐7 cells (bottom) transduced with EOS‐GFP and selected with 1 μg/mL puromycin (puro) (A), with pMXs‐Sox2‐IP or the control pBP‐GFP (B) and pLV‐tetO‐Sox2 followed by 0.5 μg/mL doxycycline (dox) treatment (C). Relative mRNA levels of TP53 and SOX2 by quantitative real‐time PCR in A‐549 cells previously transduced with empty vector (EV), sh TP53 , or sh TP53 sh SOX2 (D). Representative images and quantification of the transwell migration assay of A‐549 EV, sh TP53 , or sh TP53 sh SOX2 (E). C, control; NT, not treated. P ‐values less than 0.05 were considered statistically significant: *** p < .001; ** p < .01; * p < .05; ns not significant.

Article Snippet: To analyze the activity of regulatory regions, cells were co‐transfected with luciferase reporter plasmids PG13‐luc (wt p53 binding sites) (a gift from Bert Vogelstein; Addgene #16442) and pGL3‐Sox2 luc (a gift from Richard G. Pestell) in the presence of PEI.

Techniques: Migration, Transwell Migration Assay, Transduction, Control, Real-time Polymerase Chain Reaction, Plasmid Preparation