Fig 2: Imbalance in ROS-Glutathione Homeostasis that Blunts the DNA Damage Response by Persistent Oxidative Phosphorylation in A-iPSC and Recovery by GLUT3 Expression(A) Excessive oxidation capacity with elevated glutathione in A-iPSC and recovery by GLUT3. Glutathione analysis was conducted with the glutathione fluorometric assay. Error bars indicate SEM of three biological replicates with two independent clones in each sample group. Statistical significance was determined by two-sided t test.(B) A cellular reactive oxygen species assay kit (Abcam, ab113851) was used to measure the H2O2-scavenging activity of ESC, Y-iPSC, A-iPSC, and A-iPSC-GLUT3. Error bars indicate SEM of three biological replicates with two independent clones in each sample group. Statistical significance was determined by two-sided t test.(C) Recovery of ATM phosphorylation in A-iPSC-GLUT3 compared to A-iPSC after phleomycin treatment (2 hr, 30 µg/mL), as monitored by immunoblot in three independent representative clones.(D) In situ cell death assays of Y-iPSC, A-iPSC, Y-iPSC-shGLUT3, and A-iPSC-GLUT3 were performed 15 hr after the end of phleomycin treatment (2 hr, 30 µg/mL). Y-iPSC-shGLUT3 shows fewer cells staining for cell death compared to Y-iPSC and A-iPSC-GLUT3. The negative control is Y-iPSC treated with dye in the absence of enzymatic reaction. Nuclei are stained with DAPI. Scale bar indicates 100 µm.(E) Quantification by image analysis of apoptotic response by DNA fragmentation assay after phleomycin treatment. Error bars indicate SEM of six biological replicates with three independent clones in each sample group. Statistical significance was determined by unpaired two-sided t test.(F) Glucose uptake in Y-iPSC-shGLUT3 is reduced compared to Y-iPSC. Error bars indicate SEM of three biological replications in each sample group and two additional clones of Y-iPSC-shGLUT3. Statistical significance was determined by two-sided t test.(G) The H2O2-scavenging activity of Y-iPSC and Y-iPSC-shGLUT3. Error bars indicate SEM of five biological replicates in each sample group and three additional independent clones of Y-iPSC-shGLUT3. Statistical significance was determined by two-sided t test.(H) Excessive oxidation capacity with elevated glutathione in Y-iPSC-shGLUT3 compared with Y-iPSC. The total glutathione level was measured to determine the maximum oxidation capacity. Glutathione analysis was conducted with the glutathione fluorometric assay. Error bars indicate SEM of five biological replicates in each sample group and two additional clones of YiPSC-shGLUT3. Statistical significance was determined by two-sided t test.(I) Excessive oxidation capacity with elevated glutathione in Y-iPSC-shGLUT3. Glutathione analysis was conducted with the glutathione fluorometric assay. Error bars indicate SEM of three biological replicates with two independent clones in each sample group. Statistical significance was determined by two-sided t test.