Fig. 6.

DNA damage-inducible transcript 4 (DDIT4) induces protective autophagy against temozolomide (TMZ) cytotoxicity, but not through glucose transporter 3 (GLUT3) signaling. (A) Genes positively correlated with DDIT4 were found to be enriched in autophagy signaling using gene set enrichment analyses (GSEAs). (B) DDIT4 induced autophagy formation. After cells were transfected with 2 μg of DDIT4 plasmids for 24 h, LC3 levels from cell lysates were detected by immunoblot assays. (C) Depletion of DDIT4 reduced TMZ-enhanced autophagy formation. After transfection with 2 μg of DDIT4 shRNAs for 24 h, 200 μM TMZ was added for another 72 h. LC3 and p62 levels from cell lysates were detected by immunoblot assays. (D) Inhibition of autophagy by chloroquine (CQ) treatment reduced cell viability in DDIT4-overecpressing cells. After transfection with 2 μg of DDIT4 plasmids for 24 h, 10 μM CQ was added for another 24 h. Cell viability was measured by MTT assays. Data are the mean ± SD of 3 experiments. *p < 0.05. (E) GLUT3 showed no effects on autophagy formation. After transfection with 2 μg of GLUT3 shRNAs or cDNAs for 24 h, LC3 and p62 levels from cell lysates were detected by immunoblot assays