Figure 2

miR-27b expression induced in the differentiation stage reduced expression of mesendodermal genes. (A) Differentiation procedures. hiPS cells are seeded on Matrigel-coated plates and precultured for 1 day. Activin A (100 ng/ml) was added from day 0. hiPS cells were differentiated into mesendoderm at day 2 and definitive endoderm at day 4. (B) Expression levels of mesendodermal marker genes in hiPS-AAVS1-27b cells were analyzed by qRT-PCR in the time course of differentiation. The value of day 0 was taken as 1.0. Data are presented as mean ± SD (N = 3). The representative result from two independent experiments is shown. (C) Expression levels of differentiation marker genes in hiPS-AAVS1-27b cells were analyzed by qRT-PCR at day 2 (upper) and day 4 (lower) of differentiation. hiPS-AAVS1-27b cells were differentiated with or without dox (1 µg/ml). 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, **p < 0.01) The representative data from four independent experiments is shown. (D) Expression levels of phospho-SMAD1/5 protein in hiPS-AAVS1-27b cells at day 2 of differentiation were analyzed by Western blotting (left). Protein expression levels were quantitated and normalized by an internal reference, GAPDH (right). Original images are shown in Fig. S8A. 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 result from four independent experiments is shown.