Figure 2.
ATF4 is essential for LOP-triggered autophagy and ACD. (A,B) MZ-54 WT NHT, MZ-54 WT ATF4 KO #1 (A), MZ-54 ATG7 KO NHT and MZ-54 ATG7; ATF4 double KO (B) cells were treated with 17.5 µM LOP for the indicated time points followed by western blotting with VCL as loading control. (C) Expression levels of ATF4 mRNA were analyzed by qRT-PCR in MZ-54 WT NHT and MZ-54 WT ATF4 KO #1 cells. Fold increase of mRNA levels is shown relative to MZ-54 WT NHT with mean and SEM of 3 independent experiments performed at least in duplicate. (D) MZ-54 WT NHT, MZ-54 WT ATF4 KO #1, MZ-54 ATG7 KO NHT and MZ-54 ATG7; ATF4 double KO cells were treated with 17.5 µM or 20 µM LOP for 48 h. Cell death was assessed by measuring the propidium iodide (PI) uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. (E-H) Expression levels of ATG13 (E), WIPI1 (F), MAP1LC3B (G) and GABARAP; GABARAPL1 (H) mRNA were analyzed by qRT-PCR in MZ-54 WT NHT and MZ-54 WT ATF4 KO #1 cells treated with 17.5 µM LOP for the indicated time points. Fold increase of mRNA levels is shown relative to untreated cells of each cell line with mean and SEM of at least 3 independent experiments performed at least in duplicate. *p < 0.05, **p < 0.01, ***p < 0.001, ns: non-significant. UT, untreated