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. 2020 Aug 20;11(8):670. doi: 10.1038/s41419-020-02908-w

Fig. 6. 1,25D reduces IRX4 and NANOG expression by inhibiting TGF-β1/Smad3 signaling pathway.

Fig. 6

a The ability of 1,25D reducing IRX4 transcript at different time point was evaluated by QRT-PCR in PC-9/GR cells (mean ± SD; n = 6; ***P < 0.001). b, c The PC-9/GR cells was treated with 1,25D or gefitinib respectively for 48 h and 72 h. The expression of VDR, IRX4 and NANOG was determined by western blotting. d The percentage of CD44+CD133+ PC-9/GR cells was analyzed by flow cytometry after stimulated with TGF-β1 (20 ng/mL) for 48 h (mean ± SD; n = 3; ***P < 0.001). e The PC-9 and PC-9/GR cells were respectively stimulated with or without TGF-β1 (20 ng/mL) for 48 h, the expression of CD133 and MDR1 was detected by western blotting. f–h PC-9/GR cells were stimulated with TGF-β1 (20 ng/mL) at indicated time points, and the expression of IRX4 and NANOG was detected by QRT-PCR or western blotting. i PC-9/GR cells were stimulated with TGF-β1 (20 ng/mL) at indicated time points, the expression of p-smad3 and smad2/3 was determined by western blotting. j PC-9/GR cells were incubated with SIS3 (0.1 μM) or with their vehicle, 0.1% dimethyl sulfoxideas a control, for 1 h, and were then treated with TGF-β1 (20 ng/mL) for 48 h, the expression of IRX4 and NANOG was evaluated by western blotting. k, l PC-9/GR cells were pre‐treated with different concentrations of 1,25D for 4 h and then costimulated with 20 ng/mL TGF-β1 for 48 h. Effects of 1,25D on protein levels of p-smad3, smad2/3, IRX4 and NANOG were detected by western blotting.