Fig. 3: In a p53-null background, R273C and R273H p53 mutants abrogate canonical p53 functions and DNA binding capacity.

A LNCaP or C4-2 cells underwent CRISPR-mediated TP53 KO with subsequent, stable expression of control or mut-p53 (left). LNp53KO- and C42p53KO-mut-p53 cells were lysed and immunoblot analysis was performed with the indicated antisera (right) to show mut-p53 expression. B LNp53KO- and C42p53KO- mutp53 cells were treated with cycloheximide for the designated time points and immunoblot analysis was performed with the indicated antisera to characterize p53 stability. C Chromatin immunoprecipitation sequencing was performed in LN-pLPLUC (wt-p53), LNp53KO-R273C, and LNp53KO-R273H cells in the absence of insult. Shown are the number of p53 binding peaks (left) and binding of p53 ± 3 kb from transcriptional start sites (TSS, right). D Binding intensities of p53 peaks observed in chromatin immunoprecipitation sequencing analysis at canonical p53 target genes: CDKN1A, GADD45A, MDM2, FAS. E LNp53KO- and C42p53KO-mutp53 cells were treated with vehicle or 5 Gy IR, and 4 h post-treatment qPCR analysis of CDKN1A expression or immunoblot analysis was performed with the indicated antisera (N = 3 independent experiments). F Microarray analysis was performed in untreated LNp53KO-pLPLUC, R273C, and R273H cells. Volcano plots demonstrate differentially expressed genes in red (FC > 1.5; p < 0.05).