Extended Data Fig. 3: TP53-deleted human premalignant cells have a selective growth advantage in the setting of MNU.
a, Relative proliferation of CP-A cells treated with indicated concentrations of DCA; phase contrast images of CP-A cells treated with DMSO or 100μM DCA. b, Immunoblot showing protein levels of p53 in CP-A cells treated with DMSO or 50μM DCA. This experiment was repeated once with similar results. c, DNA content flow cytometry of CP-A cells expressing control orTP53 shRNA#2 CP-A cells treated with indicated MNU concentrations. This experiment was repeated once with similar results. d, Relative proliferation of CP-A cells treated with DMSO or indicated concentrations of MNU by Celltiter Glo. Data presented as mean ± s.d of three culture replicates; p-value calculated by two-sided Student’s t-test. e, Immunoblot showing protein levels of p53 in genetically modified CP-A cells; top panel shows protein from CP-A cells expressing control sgRNA/Cas9 or three p53 targeting sgRNAs/Cas9 after treatment with MNU 200ng/mL; bottom panel shows protein from CP-A cells expressing a scrambled or two targeting p53 shRNAs treated with DMSO, Doxorubicin, or Nutlin in the presence or absence of doxycycline. This experiment was repeated once. f, Relative proliferation of CP-A cells expressing control sgRNA/Cas9 or three p53 targeting sgRNA/Cas9 (top panel); expressing vector control, scramble control, or two p53 targeting shRNAs. Data presented as mean ± s.d of three culture replicates. g, Quantification of dsDNA breaks in CP-A cells expressing control sgRNA/Cas9 or three p53 targeting sgRNA/Cas9 in the setting of indicated concentrations of MNU by γH2AX immunofluorescence. Data presented as mean ± s.d of four technical and two cell culture replicates; p-value calculated by two-sided Student’s t-test of cell culture replicates. h, FPKM gene counts of TP53 in CP-A cells stably expressing two p53 shRNAs. i, Ratio of TP53 pathway target gene expression levels in CP-A cells expressing indicated p53 shRNA relative to scramble control (cell culture replicates shown).
