Figure 2.

Reduction of the activity of IFN signaling pathways reverses defects in cardiac differentiation of DS/CHD iPSCs

(A and B) Immunoblotting analysis of p-STAT1 and GAPDH on differentiation day 3. DS/CHD iPSCs were treated with DMSO or 1 μM JAKi from day 0 to day 3. A representative immunoblotting image is shown in A. Quantification of relative p-STAT1 levels in control cells (C62 and C68) and DS/CHD cells (D7 and D48) on differentiation day 3 in B. Each filled circle represents one independent induction, with two independent experiments for each line. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ns, not significant, ordinary one-way ANOVA.

(C) qPCR analysis of gene expression in iPSC-derived cells on differentiation day 7. DS/CHD iPSCs were treated with 1.0 μM JAKi beginning on day 0. Each filled circle represents one independent induction, with three independent inductions for each cell line. Control lines are C62 and C68 and DS/CHD lines are D7 and D49. Data are presented as mean ± SD. ∗p < 0.05, Ordinary one-way ANOVA.

(D) Representative traces of normalized GCaMP6f signal intensity showing Ca²⁺ transients in control iPSC-CMs (C68), DS/CHD iPSC-CMs (D49), and DS/CHD iPSC-CMs treated with the JAKi from day 0 to day 3. Ca²⁺ signals were imaged on day 13. Merged traces in the box are shown on right. Quantification of Ca²⁺ transient amplitude and frequency is shown in Figure S3.

(E and F) Transcriptomic analysis using RNA-seq. Three independent experiments were performed for each iPSC line (C62, C68, D7, and D48) under each condition for RNA-seq. FPKM ≥30 was set as cutoff to filter transcripts. Fold change ≥1.5 and p < 0.05 were set as criteria to determine DEGs. Venn diagrams in E demonstrating numbers of genes whose dysregulation was rescued by upon JAKi treatment on differentiation day 13. GO analysis in F of the overlapping downregulated and upregulated DEGs in DS/CHD-differentiated cells shown in E on day 13 and the rescue effects of the JAKi.