Figure 5(A) PPM1D-dependent dephosphorylation of Ulk1 at Ser637, induction of autophagy, and degradation of Noxa. PPM1D+/+ and PPM1D−/− primary thymocytes were X-rayirradiated (5 Gy), and 3 hr later, thymocytes were lysed and the expression of each protein was examined by western blotting. α-Tubulin was used as a loading control. Semiquantitative analyses are shown in Fig. EV5B. (B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (B, C) The indicated thymocytes were X-rayirradiated (5 Gy), and 3 hr later, thymocytes were immunostained with an anti-LC3 antibody. Nuclei were stained with Hoechst 33342. Representative images are shown in (B). LC3 puncta can be seen in irradiatedPPM1D+/+thymocytes (arrowheads). In (C), the proportion of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05. (B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (D) The indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were subjected to EM analysis. An autophagic vacuole (arrowhead) can be seen in an irradiated PPM1D+/+thymocyte. (B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (E) The indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, thymocytes were stained with Cyto-IDTM. Representative flow cytometry data are shown. Quantitative data are shown in Fig. EV5C. Cyto-ID fluorescence in irradiated PPM1D+/+thymocytes was higher than that in the other thymocytes. (B-F) Suppression of X-ray-induced autophagy in PPM1D−/−thymocytes. (F) Analysis of autophagic flux. The indicated thymocytes were X-rayirradiated (5 Gy) in the presence or absence of chloroquine (120 µM), and the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. Semiquantitative analyses are shown in Figure EV4D. (G-J) Contribution of PPM1D/Ulk1-dependent autophagy to irradiation-induced apoptosis. (G) Thymocytes were X-ray irradiated (5 Gy), and 6 hr later, cell death was determined using the PI assay. Data represent the mean  ± SD (n = 3 independent thymi). *p < 0.05, **p < 0.01, NS: not significant. (G-J) Contribution of PPM1D/Ulk1-dependent autophagy to irradiation-induced apoptosis. (H, I) Thymocytes were X-rayirradiated (5 Gy), and 3 hr later, the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. (I) Semiquantitative analyses are shown (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01. (G-J) Contribution of PPM1D/Ulk1-dependent autophagy to irradiation-induced apoptosis. (J) Indicated thymocytes were X-ray irradiated (5 Gy), and 3 hr later, caspase3/7 activity was examined using the Caspase-Glo 3/7 assay (Promega) according to the manufacturer's protocol. Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, **p < 0.01. (K) The effect of Noxa siRNA on thymocytes. The indicated thymocytes were transfected with the pmaxGFP vector and siRNAs. After 12 hr, cells were X-ray irradiated (5 Gy), and 6 hr or 10 hr later, the population of dead cells among the GFP-positive cells was determined using the PI assay. Data are shown as the mean  ± SD (n = 3 experiments). *p < 0.05, NS: not significant.