Figure 1.

Redox homeostasis in IDH1-mutated cells. A) Quantification of reactive oxygen species (ROS) levels by ROS-Glo H₂O₂ assay is shown for IDH1-mutated U251 cells (left panel) or doxycycline (DOX)-induced mutated-IDH1 expression U251 model (right panel). All groups were normalized to IDH1^WT dimethylsulfoxide group. N-acetylcysteine (NAC) was used as a ROS scavenger. B) Flow cytometry analysis of ROS level using CM-H₂DCFDA staining in IDH1-mutated U251 cells is shown. C) Confocal microscopy of mitochondrial ROS in IDH1-mutated cells was performed using MitoSOX staining (red). Cell nuclei were labeled with Hoechst 33342 (blue). MitoSOX signal is highlighted in the binary image (lower panel). Scale bar = 10 μm. D) Protein carboxylation analysis was done in lysate from IDH1-mutated U251 cells (upper panel). Coomassie blue staining was used as loading control (lower panel). E) Gene expression assay quantified ROS generating and scavenging genes from IDH1^WT and IDH1^R132H U251 cells. F) Immunoblot analysis for expression of NRF2 and ROS scavenging genes in IDH1-mutated glioma cells is shown. β-actin was used as loading control. G) Expression of NFE2L2 and NQO1 was measured by real-time polymerase chain reaction after 10–20 passages of DOX-induced mutated-IDH1^R132H expression in U251 cells. All groups were normalized to passage 0 group. H) Expression level of NFE2L2, HMOX1, and NQO1 were measured by real-time polymerase chain reaction after removing mutated-IDH1^R132H expression in U251 cells. All groups were normalized to DOX group. I) Cox regression analysis for the association between IDH1-mutation status and patient outcome (n = 366) is shown for lower-grade gliomas. P values were calculated using a two-sided Student t test. Error bars represent standard deviation. DMSO = dimethylsulfoxide; LGG = low-grade glioma; NAC = N-acetylcysteine.