Overexpression (OE) of miR-128a during in vitro differentiation of murine NkxCE-GFP OE-128 and OE-Ctr ESCs. (A) OE-128 and OE-Ctr ESCs (scale bars: 500 µm) were differentiated for 2 weeks (wks) by the hanging drop method (without doxycycline). Analysis was performed on respective timepoints between 0.75 and 2 wks of differentiation. Abbreviations: EB: Embryoid bodies; FACS: Flow cytometry; (B) MiR-128a was overexpressed in OE-128 EBs compared to OE-Ctr EBs from early onset of cardiogenesis (0.75 wks), becoming significant after 1 wk (p < 0.01, Mann–Whitney test); 0.75 and 1 wk: 3 assays in triplicates, different clones; 1.5 and 2 wks: 2 assays in triplicates, different clones. (C) NkxCE-GFP-positive CPCs in OE-Ctr EBs or OE-128 EBs after 1.5 wks (upper panel, scale bars: 500 µm) and after 2 wks (lower panel, scale bars: 500 µm). Images are an overlay between phase contrast and fluorescent microscopic pictures. (D) NkxCE GFP-positive CPC frequency after miR-128 OE appeared to be reduced after 1 wk, whereas the frequency of NkxCE-GFP-positive CPCs slightly increased after 1.5 wks upon miR-128 OE becoming significant after 2 wks (p = 0.0088, t-test). 1 wk: 3 assays in triplicates (different clones), 1.5 and 2 wks: 2 assays in triplicates (different clones). (E,F) Gene expression panels during NkxCE-GFP ESC OE-128 and OE-Ctr differentiation. The panels show the expression of early CPC markers after 1 wk (3 assays in triplicates, different clones) (t-tests) (E) and 1.5 wks (2 assays in triplicates, different clones) (t-tests). (F,G) The beating frequency (beats per minute, bpm) of early cardiomyocytes was significantly upregulated from 1 wk to 2 wks on mir-128a OE compared to OE-Ctr EBs (2 assays in triplicates, different clones, 5–9 videos per timepoint) (t-tests or Mann–Whitney test). Data are represented as means ± SEM. * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
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