Figure 1. ATF4 induces TMZ resistance in glioma cells.

(A) Western blot analysis of ATF4 expression in human primary astrocytes, U87 WT (wild type) cells and U251 WT (wild type) cells. (B) U87 WT cells were subjected to TMZ in a series of concentrations as indicated for 48 h, then ATF4, Nrf2 and xCT mRNA levels were evaluated by RT-PCR. n = 3, ***P < 0.001 compared with con (untreated) using one-way ANOVA. CU87 and U251 cells were transfected with ATF4 cDNA and shRNAs as described in Material and methods. ATF4 mRNA was quantified by real time RT-PCR using the ΔΔCT method with GAPDH. D, ATF4^OE and ATF4^KD U87 and U251 cells were subjected to TMZ for 3 days in a series of concentrations as indicated. The cell survival was measured by MTT assay. E, After treatment with TMZ for 3 days, the total number of vital cells was monitored. n ≥ 8 per group. Statistical analysis was performed by unpaired Student's test, *P < 0.05, **P < 0.01, ***P < 0.001, ctrl (peGFP-N1) versus ATF4-GFP or ctrl shRNA versus ATF4 shRNA.