Fig. 6 .

Glutathione rescues cells treated with carboplatin and everolimus. (A) Western blot of gamma-H2AX of pLGG cell lines treated with carboplatin or BSO for 96 hours. Numbers represent quantification of western blot expression normalized to GAPDH and vehicle. (B) Phase contrast 200× photomicrographs of cell treated with DMSO, carboplatin plus everolimus, or carboplatin, everolimus, and 4 mM glutathione for 96 hours in Res259, showing induction of cell death in combination therapy, which is prevented by glutathione supplementation. (C) Quantification of Res186 and Res259 cells that were BrdU positive by immunofluorescence 96 hours post carboplatin, everolimus, and/or glutathione treatment. (D) Quantification of Res186 and Res259 cells that were CC3 positive by immunofluorescence 96 hours post treatment. Bars represent range of samples with interquartile distribution; **P < 0.01, ****P < 0.0001 by ANOVA. (E) Cartoon of proposed mechanism in which everolimus decreases the available glutathione pool, which would normally prevent carboplatin from inducing DNA damage. Suppression of glutathione production leads to increased carboplatin-induced DNA damage and increased cell death. Scale bar is 50 microns.