Figure 5
Activated HSCs enhance chemoresistance by increasing the expression of collagen I in MCTS.
(A) LX2 cells were transfected with siCont and siCOL1A1, and their efficiency was examined by real-time PCR and (B) HepG2 cells were cultured with LX2-siCont and COL1A1-depleted LX2 cells (LX2-siCOL1A1) at a 1:1 ratio. The morphology of the spheroids was examined after 3 days. (C) Drug sensitivity was investigated in HepG2 and PLC/PRF/5 with LX2-siCont and LX2-siCOL1A1 by EthD-1 staining to detect cell death after 4 days from treating with 10 μM sorafenib. Images were obtained (left) and EthD-1 intensity was analyzed (right). Data represent the mean values ± SD from three independent experiments relative to the value for the control. (D,E) Huh7 was treated with losartan with indicated concentration for 3 days for evaluating the COL1A1 mRNA and protein levels through (D) real-time PCR and (E) western blot. Values were normalized to GAPDH. (F) HepG2:LX2 spheroids, which were co-culture at a 7:3 ratio, were treated with the indicated concentration of losartan for 3 days (upper). The volume of spheroids was analyzed (lower). Data represent the mean values ± SD from five independent experiments relative to the value for the control. (G) HepG2:LX2 spheroids, which were mixed at a 7:3 ratio, were treated with indicated concentration of losartan for 3 days and then treated with 7 μM sorafenib for an additional 4 days. Seven days after spheroid formation, the spheroids were stained with EthD-1 to detect cell death. *P < 0.05, **P < 0.005. Scale bar = 200 μm.
