Fig. 1.

Upregulation of DNA damage-inducible transcript 4 (DDIT4) expression by temozolomide (TMZ) desensitizes its cytotoxicity towards gliomas. Enhancing effects of TMZ on DDIT4 mRNA (A) and protein levels (B). After being treated with the indicated doses of TMZ for 72 h, relative mRNA and protein levels of the DDIT4 gene were analyzed by real-time PCR and immunoblot assays. Data are the mean ± SD of 3 experiments. *p < 0.05. Depletion of endogenous DDIT4 expression reduced TMZ-upregulated DDIT4 levels (C), but enhanced TMZ cytotoxicity (D). (E, F) Overexpression of DDIT4 attenuated TMZ-induced cell apoptotic death and caspase-3 activity. (G, H) Knockdown of DDIT4 enhanced TMZ-induced caspase-3 activation. After cells were transfected with 2 μg cDNA or shRNA for 24 h. Then, 200 μM of TMZ were added for another 72 h. DDIT4 and cleavage caspase-3 (Cle-Casp 3) protein levels were detected by immunoblot assays. Caspase-3 activity was measured by using commercial kit according to the manufacturer’s instructions listed in method section. Cell viability was measured using an MTT assay. Data are the mean ± SD of 3 experiments. *p < 0.05