Supplementary Figure 4: Recovery of ROS-glutathione homeostasis in A-iPSC by reduction (shRNA-GSS) of excessively activated GSS and disruption of ROS-glutathione homeostasis in Y-iPSC by overexpression of GSS.
From: ZSCAN10 expression corrects the genomic instability of iPSCs from aged donors

(a) Expression levels of GSS after shRNA knockdown of ZSCAN10 in Y-iPSC. The red dashed line denotes data that supports the findings presented in the manuscript (Fig. 2e). Error bars indicate standard error of the mean of two technical replicates with three independent clones in each sample group (n = 3). Statistical significance was determined by two-sided t-test. (b) Confirmation of the ZSCAN10 binding activity to the GSS promoter that reported by Yu et al., J. Biol. Chem., 2009 using ChIP–qPCR14. Oct4 promoter region was used as a positive control and an 80-bp genomic region on chromosome 4 was used as a negative control. Error bars indicate standard error of the mean of five replicates for AiPSC and YiPSC (n = 5) and three replicates for positive/negative controls (n = 3). Statistical significance was determined by two-sided t-test. Detailed information provided in Supplementary Table 5. (c) Expression levels of GSS after shRNA knockdown of GSS in A-iPSC. Error bars indicate standard error of the mean of two technical replicates with three independent clones in each sample group (n = 3). Statistical significance was determined by two-sided t-test. Detailed information provided in Supplementary Table 5. (d) Oxidation capacity of glutathione in A-iPSC, A-iPSC-shRNA-GSS, Y-iPSC, and Y-iPSC-GSS. Quantification of reduced glutathione (GSH) and oxidized glutathione (GSSG) was measured to determine the total glutathione level (maximum oxidation capacity). Glutathione analysis was conducted with the Glutathione Fluorometric Assay (Biovision, K264-100). Error bars indicate standard error of the mean of two technical replicates with four independent clones in A-iPSC-shRNA GSS (n = 4) and two technical replicates with seven independent clones in A-iPSC-shRNA GSS (n = 7). Statistical significance was determined by two-sided t-test. (e) H2O2 scavenging activity in ESC, Y-iPSC, Y-iPSC-GSS, A-iPSC, A-iPSC-ZSCAN10, and A-iPSC-shRNA-GSS. A cellular reactive oxygen species assay kit (Abcam, ab113851) was used to measure the H2O2 scavenging activity. Excessive H2O2 scavenging activity in A-iPSC was recovered by shRNA-GSS expression. The excessive H2O2 scavenging activity seen in A-iPSC was recapitulated by GSS overexpression in Y-iPSC. Mean ± standard deviation is plotted for four replicates from each condition. Error bars indicate standard error of the mean of three biological replicates with three independent clones in Y-iPSC-GSS and A-iPSC-shRNA GSS (n = 9). Statistical significance was determined by two-sided t-test. The red dashed line denotes data that supports the findings presented in the manuscript (Fig. 3f and 3 h). (f) Expression levels of GSS in Y-iPSC after overexpression of GSS. Error bars indicate standard error of the mean of two technical replicates with three independent clones in each sample group (n = 3). Statistical significance was determined by two-sided t-test.