Fig. 1

Establishment of H1-KO, H2-KO and H1/H2-dKO hESC lines. A Schematic diagram of cardiomyocyte differentiation protocol. Mesoderm stage was at day 2. Cardiac mesoderm stage was at day 3. Cardiac progenitor stage was at day 5. Cardiomyocytes started beating at day 7. RB-: RPMI/B27-, RB: RPMI/B27, CHIR: CHIR99021, Vc: Vitamin C. B Temporal expression of HAND1 and HAND2 during cardiomyocyte differentiation. Relative to GAPDH expression (n = 3). C Schematic diagram of CRISPR/Cas9 mediated HAND1/2 genome editing. PAM: protospacer adjacent motif. D Expression of OCT4, SOX2 and NANOG in WT and HAND KO hESC lines. Relative to GAPDH expression (n = 3). E Immunofluorescence staining of SSEA-4 and OCT4 in WT and HAND KO hESC lines. Scale bar = 100 μm. F Flow cytometry analysis of SSEA-4⁺ cells in WT and HAND KO hESC lines. CTRL represented the negative control with secondary antibody incubation only. G qPCR analysis of HAND1 and HAND2 in WT and HAND KO hESC lines-derived day 10 cardiomyocytes. Relative to GAPDH expression (n = 3). One-way ANOVA. H Western blot analysis of HAND1 and HAND2 expression in WT and HAND KO hESC lines-derived differentiation day 10 cardiomyocytes. GAPDH served as loading control. Corresponding uncropped full-length gels and blots are presented in Additional file 8: Fig. S7. **p < 0.01, ***p < 0.001, ****p < 0.0001