Fig. 2. ATF4 is required for the apoptotic response upon dual inhibition of MDM2 and PPM1D.

a ATF3 and ATF4 mRNA induction in cell lines treated with vehicle (0.2% DMSO), 10 μM nutlin-3a, 25 μM GSK2830371, or both drugs for 24 h. q values were calculated by the DESeq2 software. b p53 occupancy at ATF3 and ATF4 gene loci analyzed by ChIP-seq. c Transcriptional activity at ATF3 and ATF4 gene loci measured by GRO-seq in cell lines treated with 10 μM nutlin-3a for 60 min. d Western blots in TPC1 and K1 cell lines treated with indicated compounds for 6 h. Results shown here are representative of three independent experiments. e, f TPC1 cells depleted of ATF3 or ATF4 were treated for 72 h, stained with Annexin V-FITC/PI and analyzed by flow cytometry. Statistically significant difference was calculated by paired, two-sided t test, n = 4 (e), n = 3 (f) independent experiments. g TPC1 cells transduced with Tet-on-ATF4 expression vector were treated with 10 μg/ml doxycycline, vehicle control, or drug combination for 72 h prior to flow cytometry measurement of Annexin V-FITC/PI positive cells. Paired, two-sided t test was used for calculations of statistical significance (n = 3 independent experiments). h, i Q-RT-PCR of p53 target genes in the TPC1 cell line treated with the indicated compounds for 24 h. Paired, two-sided t test was used to calculate the indicated p value, n = 3 independent experiments. Data in e–i are represented as mean ± SD. j ATF4 occupancy analyzed by chromatin immunoprecipitation (ChIP) followed by Q-PCR at p53 target genes. See also Supplementary Figs. 2 and 3. Source data are provided as a Source data file.