Fig. 6

Direct targeting of TET2 by miR-20b-5p during cardiac differentiation of hESCs. A qRT-PCR analysis of the relative TET2 mRNA expression level during 12-day cardiac differentiation of hESCs with exposure to m-NC (mimic-negative control), miR-20b-5p-mimic, i-NC (inhibitor-negative control) or miR-20b-5p-inhibitor (n = 3). B and C Western blot analysis B and quantification C of the relative TET2 protein level in 12-day cardiac-differentiated hESCs with exposure to m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor (n = 3). D and E Dot blot analysis D and quantification E of 5hmC expression in 12-day cardiac-differentiated hESCs with exposure to m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor (n = 4). Methylene blue staining demonstrated equal loading. F and G Flow cytometry analysis G and quantification F of TET2-positive cells in 12-day cardiac-differentiated hESCs with exposure to m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor (n = 3). H Immunofluorescence staining of TET2 in 12-day cardiac-differentiated hESCs with exposure to m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor (Scale bar = 10 μm). I The dual luciferase reporter system was utilized to construct the predicted binding sequences of miR-20-5p to the 3’ untranslated region of TET2 (LUC-WT-TET2) and a corresponding mutated sequence (LUC-MUT-TET2). J and K The relative luciferase activity was detected in HEK-293 T cells transfected with LUC-WT-TET2 or LUC-MUT-TET2 with exposure to m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p inhibitor (n = 3). D3, D6, D9 and D12 indicated the time points at Day 3, Day 6, Day 9 and Day 12, respectively. Quantitative data were presented as mean ± SEM. 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, while n.s. indicated non-significance