Fig. 3.

Effect of TMZ and a TRPA1 agonist and inhibitor on antioxidant gene expression. TMZ and PF-4840154 cotreatment prevented the effect of TMZ on antioxidant gene expression. Pretreatment with HC030031 or HC030031 and TMZ cotreatment significantly increased MnSOD mRNA expression. The fold change in mRNA expression of MnSOD (A, F), NQO1 (B, G), and heme oxygenase (HO)-1 (C, H) in U251 and SHG-44 cells as measured by quantitative RT–PCR. The expression of GSH were increased by exposure to TMZ and decreased in PF-4840154 and TMZ cotreatment group, as detected in U251 cells (D) and SHG-44 cells (I) with a fluorescence plate reader. The levels of mitochondrial ROS were increased by exposure to PF-4840154 and TMZ cotreatment, as detected by MitoSOX Red fluorescence in U251 cells (E) and SHG-44 cells (J) with a fluorescence plate reader. Intracellular ROS levels in in U251 cells and in SHG-44 cells were detected by DCFH-DA and fluorescence microscopy with a fluorescence plate reader (K). Representative microscopy images are shown (100 x) with mean values and data from individual plate reader experiments reported graphically below. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001