Figure 3

miR-27b antagonized the BMP signaling pathway during early differentiation. (A) hiPC-AAVS1-27b cells were differentiated into mesendoderm as in Fig. 2A with or without dox (1 µg/ml). Protein levels of phospho-SMAD1/5 were analyzed every 12 h of mesendoderm differentiation by Western blotting. Original images are shown in Fig. S8B. The representative result from two independent experiments is shown. (B) hiPC-AAVS1-27b cells were differentiated into mesendoderm as in Fig. 2A with or without dox (1 µg/ml). Expression levels of direct target genes of BMP signaling were analyzed by qRT-PCR. The value of dox- was taken as 1.0. Data are presented as mean ± SD (N = 3). Student’s t test was performed (**p < 0.01). The representative data from three independent experiments is shown. (C) hiPC-AAVS1-27b cells were differentiated into mesendoderm as in Fig. 2A with or without dox (1 µg/ml) and BMP4 (10 ng/ml), and mesendodermal marker genes were analyzed by qRT-PCR. The value of BMP-dox- was taken as 1.0. Data are presented as mean ± SD (N = 3). One-way ANOVA was performed, followed by Tukey’s post-hoc test. Groups labeled ‘b’ are significantly different from labeled ‘a’ (p < 0.05). The representative data from two independent experiments is shown. (D) Gene expression levels in EBs were analyzed 3 days after seeding by qRT-PCR. Dox (1 µg/ml) was added starting from 24 h after seeding. The value of dox- was taken as 1.0. Data are presented as mean ± SD (N = 3). Student’s t test was performed (*p < 0.05) The representative data from four independent experiments is shown.