Fig. 2

FXR inhibits Notch1 expression in HCC cells and directs the asymmetric division of Sox9⁺ liver CSCs. a Huh7 cells were treated with CDCA (100 μM) or DMSO for 24 h, and the expression of SHP and Notch1 was examined by quantitative real-time PCR. b Huh7 cells were treated with GW4064 (10 μM) or DMSO for 24 h, and the expression of SHP and Notch1 was assayed by quantitative real-time PCR. GAPDH was used as an internal control for the examination of SHP and Notch1. c Effects of FXR agonists (CDCA or GW4064) on Notch1 protein expression. Huh7 cells were treated with CDCA (100 μM) or GW4064 (10 μM) for 36 h. Total protein samples were collected and subjected to Western blotting analysis to detect Notch1 protein expression, normalized to GAPDH. d the schematic depiction shown the BrdU pulse-chase and paired-cell assays. e Left: Representative images of the paired-cell assay in CD133⁺ liver CSCs from Huh7 cells. BrdU, red; DNA, blue; Sox9, green. Scale bars: 5 μm. Right: Quantification of BrdU asymmetry or symmetry in Sox9⁺ liver CSCs maintaining in 10% stripped FBS-containing medium supplemented with GW4064 (10 μM) or DMSO for 24 h. f Western blotting was used to detect the protein levels of NICD1 and Numb in Huh7 cells maintaining in 10% stripped FBS-containing medium supplemented with GW4064 (10 μM) or DMSO for 36 h. Data were presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001