Fig. 5

Expression of NANOG in response to cytokine inhibitors and hBMP4. A Optical and fluorescence microscopy showing NANOG expression in NANOG tdTomato knock-in positive cells grown in the presence of cytokine inhibitors. (a) Light microscopy showed that NANOG tdTomato PC-iPS cells were dome-shaped in all the groups. Scale bar 200 μm. (b) Fluorescence microscopy showed that only SB431542 detectably inhibited NANOG tdTomato expression. Scale bar 200 μm. (c) Flow cytometric analysis of NANOG tdTomato knock-in positive cells cultured in the presence of inhibitors. (d) RT-PCR analysis of RNA harvested from NANOG tdTomato knock-in positive cells cultured in the presence of inhibitors. B Flow cytometric and RT-PCR showing NANOG expression in NANOG tdTomato knock-in positive cells grown in the presence of BMP4 and Noggin. (a) Light microscopy showed that most of the cells treated with 10 ng/ml hBMP had differentiated, but cells in the control and antagonist groups had not. Scale bar 200 μm. (b) Fluorescence microscopy also showed that cells treated with 10 ng/ml hBMP had markedly lower expression of NANOG tdTomato, compared with cells cultured in T1 alone and cells receiving antagonists. Scale bar 200 μm. (c) Flow cytometric analysis of NANOG tdTomato knock-in positive cells cultured in the presence of BMP4 and Noggin. (d) RT-PCR analysis of RNA harvested from NANOG tdTomato knock-in positive cells cultured in the presence of BMP4 and Noggin. ****< 0.0001; ***< 0.001; **< 0.01, *< 0.5; ns, not significant