High-Fidelity Reprogrammed Human IPSCs Have a High Efficacy of DNA Repair and Resemble hESCs in Their MYC Transcriptional Signature

Figure 5

C-MYC maintained high-quality and high-efficiency NHEJ and is required for less error-prone DSB repair. (a)(i) Western blot analysis from whole cell extracts of H9 treated with either solvent control (DMSO) or MYC inhibitor (10058-F4) for 24 h at 50 μM, exposed to IR (1 Gy), and collected at indicated time points. (a)(ii) Densitometry analysis comparing the means from three independent western blots as in ((a)(i)). Statistical significance of the data was determined using 2-way ANOVA with Bonferroni posttests (γH2AX is significantly different between DMSO and MYC inhibition at 1 h, 2 h, and 4 h, ; pATM is significantly different at 1 h, ; Ku80 is significantly different at 0 h and 2 h, ). ((b)(i) and (b)(ii)) The graph represents (i) efficiency of end-joining repair and (ii) percentage of misrepair in linearized PUC18 (with compatible ends) following incubation with extracts from H9 cells MYC siRNA. Repair efficiency is calculated by counting the total number of colonies (correctly repaired (blue) + incorrectly repaired (white)) from in vitro assays. Statistical significance was determined using paired -test analysis ( between data sets H9 versus H9 siMYC). (c) Shown is the heatmap of log₂ mean-subtracted normalized values of signal intensities from averaged independent biological replicate microarray samples (–6 per condition) representing the expression of genes in MYC module in mRNA from donor fibroblasts, donor CB (CD34⁺ population), and CB-iPSC lines (i.e., CB.iPS + MSC and CB.iPS (minusMSC)). ((d)(i) and (d)(ii)) (i) Shown is a representative gel image of the PCR products recovered from CB6.2 and iCB9 with or without treatments with siMYC. The PCR products are either redigested with I-Sce1 or left uncut (U). (S−) products represent the I-Sce1 resistant repair products. These (S−) fragments are cloned into TOP10 competent cells. (ii) The clones, each representing different repair products, were analyzed by sequencing near I-Sce1 junction. Data represents ~10–15 clones analyzed in H9, CB6.2, iCB9, and iHuF3. The data is significantly different () for CB6.2 versus CB6.2 siMYC (>20 nt deletion) and iCB9 versus iCB9 siMYC (>20 nt deletion). Results are representative of the mean of two independent experiments of each set ± SEM; , , , and , based on -test analysis.

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