Expression of tRNAiMet controls glioblastoma cell growth. (A) Northern blotting analysis showing down-regulated expression of tRNAiMet by two separate targeting sgRNAs in U251 glioblastoma cells. A sgRNA targeting GFP was used as a control. The expression of 5S rRNA was used as loading controls. (B) Confocal images of proliferating U251 glioblastoma cells after transduction of virus expressing the indicated sgRNAs. Virus-transduced cells are identified by the coexpressed mCherry at 7 dpi. (Scale bar for lower magnification images, 50 µm.) (C) Quantification of the effect of knockdown of tRNAiMet on cell proliferation at 7 dpi (mean ± SEM; n = 3; ****P < 0.0001). (D) A time-course analysis of U251 cell proliferation by measuring ATP-dependent luminescence (mean ± SEM; n = 6; *P < 0.05, **P < 0.01, and ****P < 0.0001). (E) Knockdown of tRNAiMet greatly reduces proliferation of human GSCs. Cell proliferation was measured by ATP-dependent luminescence for three individual human GSC isolates (GSCs-1, GSCs-2, and GSCs-3; mean ± SEM; n = 6; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).