Fig. 6. Loss of Becn1 minimal affected early chromatin accessibility of iCMs but altered transcriptomic profile at later stage.

(A) Signal intensity (top) and heatmap (bottom) of ATAC-seq signals in cells treated with LacZ + shNT, LacZ + shBecn1, MGT + shNT, and MGT+shBecn1 and collected 3 days after viral transduction. Average reads from a 2-kb region across the transcriptional start sites (TSSs) were used. The color in heatmap represents the intensity of chromatin accessibility. (B) Differential analysis of ATAC-seq peaks between LacZ versus MGT or shNT versus shBecn1 samples. Yellow indicates LacZ-transduced shNT or shBecn1 cells, and green indicates the MGT-transduced cells. Two-way arrows indicate the samples for differential analysis. The number of differential regions is shown. Gain, numbers of sites that gain chromatin accessibility; Loss, numbers of sites that lose chromatin accessibility. (C) Hierarchical clustering and heatmap showing the expression of cardiac and fibrotic genes in samples as in (A) at reprogramming day 3 and day 5. (D and E) GSEA analysis shows enrichment of indicated gene sets in MGT + shBecn1 cells compared to MGT + shNT cells on reprogramming day 3 (D) and day 5 (E). (F and G) Overlap of up-regulated (F) band down-regulated (G) differentially expressed genes (DEGs) induced by Becn1 knockdown between MGT and LacZ-transduced cells on reprogramming day 5. (H and I) GO analysis of up-regulated (H) and down-regulated (I) DEGs using gene list from (F) and (G), respectively. (J) qPCR analysis of indicated gene expression at different time points of reprogramming. All experiments were repeated at least three times. Mean values from technical triplicates were used for statistics. Two-tailed unpaired t test was used for (J) comparing shBecn1 versus shNT at each time point. Error bars indicate means ± SEM; **P < 0.01 and ***P < 0.001.