Fig. 7.

OGM induces ferroptosis through upregulation of ATF4. (A) CRISPRi knock-down of ATF4 prevented OGM-induced cell death in U87 cells, while guide RNAs or dCas9 alone had no effect on survival. (B) Knock-down of ATF4 prevented OGM-induced cell death in U138 cells. (C) CRISPRi successfully reduced ATF4 expression even in the setting of OGM-induced expression in U87 cells and (D) in U138 cells. (E) CRISPRi mediated knock-down of ATF4 prevented OGM-induced expression of ferroptosis marker TFRC in U87 cells and (F) in U138 cells. (G) CRISPRi knock-down of ATF4 prevented OGM induced expression of direct ATF4 target CHAC1 in U87 cells and (H) in U138 cells. (I) CRISPRi knockdown of ATF4 prevented OGM-induced increase in oxidative stress response marker HMOX1 in U87 cells and (J) in U138 cell. (K) Model depicting effects of OGM in GBM cells. Lactic acid accumulation from the Warburg effect activates GPR68, which suppresses ATF4 transcription. GPR68 inhibition by OGM induces ATF4 expression, which then increases CHAC1, leading to depletion of glutathione. This ultimately causes accumulation of toxic lipid peroxides, which triggers ferroptosis