Fig. 3. ATF4 accumulation upon dual MDM2/PPM1D downstream of heme depletion, HRI induction, and eIF2α phosphorylation.

a Schematic of signaling pathways leading to inhibitory phosphorylation of eIF2α. The key residue mediating this inhibitory phosphorylation is serine 51. Created with BioRender.com. b Western blots of cells treated with vehicle (0.2% DMSO), 10 μM nutlin-3a, 25 μM GSK2830371, or the drug combination for indicated times. p-eIF2α indicates S51 phosphorylation. c Cells treated with indicated compounds for 24 h were lysed and subjected to polysome profiling by using sucrose density gradient fractionation. d Polysome to monosome ratios. Absorbances displayed in c were quantified, and statistical significance (n = 3 independent experiments) was calculated using paired, two-sided t test. e Western blots in cells treated with indicated compounds for 24 h. f Western blots in cells transduced with non-targeting shRNA controls (shCTRL) or two different shRNAs targeting HRI (EIF2AK1). p-eIF2α indicates S51 phosphorylation. g, k TPC1 cells depleted of HRI (g) or HMOX1 (k) were treated with vehicle control or the drug combination for 72 h. Fraction of apoptotic cells was determined by flow cytometry. Statistical significance (n = 3 independent experiments) was calculated by paired, two-sided t test. h, j Cellular levels of free (regulatory) heme were measured in cells treated with denoted compounds for 6 h. Paired, two-sided t test was used for calculations of statistical significance (n = 3 independent experiments). i Western blots in cells treated with the indicated compounds for 24 h. l A schematic of HRI activation upon dual inhibition of MDM2 and PPM1D. Created with BioRender.com. Results shown in b, e, f, i are representative of three independent experiments. Data in d, g, h, j, k are represented as mean ± SD. See also Supplementary Figs. 4 and 5. Source data are provided as a Source data file.