Fig. 4

Prolonged treatment of HCC cells with PI3K inhibitors leads to activation of SGK3 and expansion of liver CSCs. a MHCC97H cells were treated with either LY294002 (concentration of 2.5, 5,10, 20, 40 μM) or ZSTK474 (concentration of 0.5, 1, 2.5, 5, 10 μM) for 24 h. Dimethyl sulphoxide (DMSO) was used as the control. Cell lysates were subjected to western blot analysis with the indicated antibodies. b Expression of CD133 was detected by RT-PCR in MHCC97H cells treated with ZSTK474 for 24 h. c Expression of Nanog was detected by RT-PCR in MHCC97H cells treated with ZSTK474 for 24 h. d Huh7 and MHCC97H cells were treated with 5 μM of ZSTK474 for 0 to 72 h. Cell lysates were subjected to western blot analysis with the indicated antibodies. e The proportion of CD133+ cells in Huh7 cells was evaluated by flow cytometric assay. f The suppression of SGK3 by siRNA in MHCC-97H cells treated with 5 μM of ZSTK474 resulted in a rescue of CD133 and Nanog overexpression, detected by RT-PCR. g The effect of the Class I PI3K inhibitor ZSTK474 on the growth of MHCC97H cells. h The protein levels of Nanog, CD133, pSGK3 and SGK3 in MHCC97H tumour xenografts in control and ZSTK474-treated mice. i The mRNA levels of CD133 in MHCC97H tumour xenografts in control and ZSTK474-treated mice. j Immunohistochemical staining of CD133 in the xenografted tumour xenografts treated with ZSTK474 or control. Scale bars, 20 μm. *P < 0.05