Fig. 5

Inhibition of hESC-derived cardiac differentiation by miR-20b-5p. A-C qRT-PCR analysis of miR-20b-5p level during 12-day cardiac differentiation in hESCs-CMs A, hiPSCs-CMs B and mESCs-CMs C (n = 3–4). D qRT-PCR analysis of miR-20b-5p level in embryonic and neonatal heart samples (E17.5, P2 and P9) from wild-type C57BL/6 mice (n = 3). E qRT-PCR analysis of the relative mRNA expression level of cardiac transcriptional factors (GATA4, NKX2.5, TBX5, MYH6 and cTnT) in 12-day cardiac-differentiated hESCs with treatment of m-NC (mimic-negative control), miR-20b-5p-mimic, i-NC (inhibitor-negative control) or miR-20b-5p-inhibitor (n = 3–5). F and G Western blot analysis G and quantification F of the relative protein levels of cardiac transcriptional factors (GATA4, NKX2.5, TBX5, MYH6 and cTnT) in 12-day cardiac-differentiated hESCs with treatment of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor (n = 3). H Immunofluorescence of cTnT in 12-day cardiac-differentiated hESCs with treatment of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor (Scale bar = 10 μm). I and J Flow cytometry analysis I and quantification J of cTnT-positive cells in 12-day cardiac-differentiated hESCs with treatment of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor (n = 3). D0, D3, D6, D9 and D12 indicated the time points at Day 0, Day 3, Day 6, Day 9 and Day 12, while E17.5, P2 and P9 indicated Embryonic day 17.5, Postnatal day 2 and 9, respectively. Quantitative data were presented as mean ± SEM, while statistical significance was analyzed via a one-way ANOVA followed by Bonferroni multiple comparisons test and represented as *P < 0.05, **P < 0.01 and ***P < 0.001