Supplementary Figure 6: Higher somatic cell ROS among the tissue donors as a causative origin of the genomic instability in A-iPSC and recovery by glutathione treatment in the early stage of A-iPSC reprogramming. | Nature Cell Biology

Supplementary Figure 6: Higher somatic cell ROS among the tissue donors as a causative origin of the genomic instability in A-iPSC and recovery by glutathione treatment in the early stage of A-iPSC reprogramming.

From: ZSCAN10 expression corrects the genomic instability of iPSCs from aged donors

Supplementary Figure 6

(a,b) Somatic cell ROS measured by MitoSOX staining. Mitochondrial Superoxide Indicator, MitoSOX Red dye (ThermoFisher, M36008) was used to measure somatic cell ROS from young somatic cells (Y-SC) from B6CBA mouse and aged somatic cells (A-SC) from B6129 and B6CBA mice (a), and human young somatic cells (Y-SC) from MRC5 donor and human aged somatic cells (A-SC) from LS and AG4 donors (b). Reduced level of the somatic cell ROS with the treatment of the stabilized form of glutathione chemical (3 mM of glutathione reduced ethyl ester, CAT #G-275-500, GoldBio) for three days in the media in A-SC from AG4 donor (A-SC-AG4-glutathione) (b). Scale bar indicates 100 μm. (c,d) Quantification of the MitoSOX staining (ROS) level using image-based quantification (ImageJ software) from the samples in Supplementary Fig. 6A, B. Error bars indicate standard error of the mean of independent colonies (n = 10). Statistical significance was determined by unpaired two-sided t-test. (ef) Representative phenotypes of the reprogrammed iPSC from the donor somatic cells from Supplementary Fig. 6A, B from mouse and human donors. A-SC-AG4 somatic cells with the reduced ROS by the treatment of the glutathione reduced ethyl ester in Supplementary Fig. 6B, D was studied. (g) Immunoblot of pATM showing that A-iPSC with glutathione treatment recover the DNA damage response in the biologically independent clones after phleomycin treatment. A-iPSC were generated with the treatment of 3 mM glutathione reduced ethyl ester prior to and during the early stage of reprogramming (from one day before reprogramming virus infection to 10 days post reprogramming virus infection). (h) Copy number profiling analysis of human A-iPSC with glutathione treatment from 10 clones (upper panel). Schematic diagrams represent 10 non-rearranged A-iPSC with glutathione treatment, compared with seven rearranged A-iPSC from 11 clones (lower panel) without glutathione treatment. A-hiPSC (n = 11 (7/11), p = 0.64) and A-hiPSC-glutathione (n = 10 (0/10), p < 4E-5). The p values are the observed (p) and estimated likelihoods (p) of detecting no rearrangements in the absence of lineage effects using a binomial distribution, respectively. (I) qPCR of ZSCAN10. Error bars indicate standard error of the mean of two replicates with two independent clones in each sample group in Supplementary Fig. 6G. Statistical significance was determined by two-sided t-test (n = 4). (J) qPCR of GSS. Error bars indicate standard error of the mean of two replicates with two independent clones in each sample group in Supplementary Fig. 6G. Statistical significance was determined by two-sided t-test (n = 4).

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