Fig. 5: Impaired p53-dependent gene expression and DNA damage responses in DPF2 R350H mutant cells.
a Doxorubicin-induced expression of p53. DPF2 WT and R350H (H/H) HCT116 cells were treated with 0.5 μM doxorubicin for 24 h, and protein levels were analyzed by immunoblotting. Data are representative of two independent experiments. b Venn diagram showing the overlap of genes upregulated ( > 1.5-fold, FDR < 0.05) by doxorubicin treatment in WT cells and genes whose doxorubicin-induced upregulation in H/H cells was reduced to less than 0.5-fold compared with that in WT cells (FDR < 0.05). c Box plot displaying expression levels of the 75 overlapping genes in b. The box plots indicate the median (central line), the third and first quartiles (box edges), and 1.5 × interquartile range (IQR) above and below the box (whiskers) (n = 1 as biological replicates were combined). P-values were determined by two-tailed unpaired Student’s t-test. TPM, transcripts per million. d KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis of the 75 overlapping genes in b. The Fisher’s exact test p-values were obtained using the GeneSCF functional enrichment tool. e mRNA levels of p53-dependent p21, NOXA, KAI-I, and PAI-I genes, with or without 0.5 μM doxorubicin treatment for 48 h, were measured by RT-qPCR and normalized to that of TBP mRNA. The levels of transcription of each gene in WT cells without doxorubicin treatment were defined as 1. Data are presented as mean values ± SD of three independent experiments. Cell-viability (f), colony-formation (g), and caspase-activity (h) assays using p53-/- (control) and DPF2 WT and H/H HCT116 cells. Cells were treated with different concentrations (0, 100, 200, 400, 800, and 1000 nM) (f, g) and 1000 nM (h) of doxorubicin. Data are presented as mean values ± SD of six independent experiments (f, h). Data are representative of three independent experiments (g). P-values were determined by two-tailed unpaired Student’s t-test (e, h). Source data are provided as a Source Data file.
