Extended Data Fig. 1: ME2, a non-target gene of mutant p53, affects mutant p53 transcriptional activity.
From: Malic enzyme 2 maintains protein stability of mutant p53 through 2-hydroxyglutarate
a, Related to Fig. 1a. SF188 cells were transfected with control siRNA (siCtrl) or siRNA against ME1, ME2, and/or p53 for 48 hours, and RNA were extracted for real-time PCR analysis of target knockdown efficiency and expression of p53 (n = 3 biological independent samples). b, Reverse sequencing analysis of mutation(s) in TP53 gene in SF188 cells. Blue shading indicates the mutation position. A GGA-to-GAA mutation has been detected in TP53 gene in these cells. c and d, SF188 cells transduced with lentivirus expressing sgRNA targeting p53 or sgRNA only (Ctrl) were transfected with control siRNA (siCtrl) or siRNA against ME1, or ME2 for 48 hours, and RNA were extracted for real-time PCR analysis of expression of indicated genes (n = 3 biological independent samples). e and f, HCT116 cells and U2OS cells were transfected with Ctrl or p53 siRNA (sip53) for 48 hours. mRNA and protein expression were determined by quantitative RT-PCR (e) and western blotting (f) respectively. n = 4 biological independent wells. g and h, U251MG cells, SF188 cells and MDA-MB-231 cells were transfected with Ctrl or p53 siRNA for 48 hours. mRNA and protein expression were determined by quantitative RT-PCR (g) and western blotting (h) respectively. n = 4 independent wells. i-k, HEK293 cells and p53-null H1299 cells were transiently transfected with different mutant p53 cDNAs individually or vector control plasmid (-) as indicated for 48 hours. mRNA and protein expression were determined by quantitative RT-PCR (i, j) and western blotting (k) respectively. n = 4 independent wells. Data in a, c, d, e, g, i, j are means ± SD, P values were determined by unpaired two-tailed Student’s t-tests. All western blot data in this study are representative of three independent experiments unless otherwise noted.
