Figure 2. TMZ induces cell death in an ATF4-dependent manner.

(A and B) ATF4^OE and ATF4^KD U87 cells were treated with TMZ at indicated concentrations for 3 days. Cell death was demonstrated by propidium iodide (PI) staining. Scale bar represents 100 μm. n ≥ 3 per group. Statistical analysis was performed by unpaired Student's test. ***P < 0.001, ctrl (peGFP-N1) versus ATF4-GFP or ATF4 shRNA versus ctrl shRNA. (C) Visualization of ATF4^OE and ATF4^KD U87 cells treated with TMZ for 3 days. Scale bar represents 100 μm. (D) Cell death analysis was performed by flow cytometer with 7-AAD (late apoptosis) and Annexin V (early apoptosis) staining. (E) Quantification of apoptotic cell death in ATF4^OE and ATF4^KD U87 cells treated with TMZ. n = 3 per group. Statistical analysis was performed by unpaired Student's test. **P < 0.01, ***P < 0.001, ctrl versus ATF4-GFP or ctrl shRNA versus ATF4 shRNA; ^###P < 0.001, ATF4-GFP versus ATF4 shRNA.