Fig. 4

MTHFD2 expression provides survival advantage to GBM cells from glutamine deprivation. See also Additional File 2: Supplemental Fig. 4. a, b U87 cells were transfected with two types of MTHFD2 siRNA and scrambled control siRNA constructs for 24 h and changed to medium with or without glutamine at Day 1. Cell number over time represents the mean ± SEM of three independent experiments (statistically significant with **p < 0.01). Immunoblot images of MTHFD2 and actin were obtained from cell lysate. c Representative images of U87 cells with TUNEL staining. Scale bar: 100 μm. Cells were transfected with siRNA constructs against MTHFD2 and control LacZ which were grown with or without glutamine for 48 h. Quantification of TUNEL-positive cells was performed with the ImageJ analysis. Data represent the mean ± SEM of three independent images for each group (statistically significant with **p < 0.01). d NAD +/NADH ratio in U87 cells transfected with siRNA constructs against MTHFD2 and control LacZ which were grown with ± glutamine and ± NADH 1 mM for 48 h. Data represent the mean ± SEM of three independent experiments (statistically significant with **p < 0.01). e Representative fluorescence microscopy images of ROS signal in U87 cells. Scale bar: 100 μm. ROS measurement in U87 cells transfected with siRNA constructs against MTHFD2 and control LacZ which were grown with ± glutamine for 48 h. ROS signal was inhibited by an antioxidant, 50 mM N-acetyl cysteine (NAC). Data represent the mean ± SEM of three independent experiments (statistically significant with *p < 0.05, **p < 0.01)