Fig. 5
Activin-A–ALK4 axis contributes to the maintenance of CIC-like cells but not to the spheroid formation of MSTO-211H cells. a MSTO-211H cells were cultured for 4 days under sphere-forming conditions in the presence of normal mouse IgG (control) or anti-activin-A monoclonal antibody (10 μg/ml), and the spheroid formation was analyzed. b ALDHbright cells in MSTO-211H spheroids formed in the presence of normal mouse IgG (control) or anti-activin-A monoclonal antibody (10 μg/ml) was determined by ALDEFLUOR assay. c, d shRNA-transfected MSTO-211H cells (shLMNA, shALK4-1, or shALK4-2) were cultured for 4 days under sphere-forming conditions. The expression of ALK4 on MSTO-211H was knocked down using shRNA (shALK4-1, shALK4-2). c The spheroid formation was analyzed. d Total RNA was isolated, and the relative expression of ALDH1a1 was determined by real-time PCR. Each mRNA level was normalized to the endogenous GAPDH. e MSTO-211H cells were cultured for 4 days under sphere-forming conditions in the presence of DMSO (control) or ALK inhibitor, SB431542 (10 μm) and the spheroid formation was analyzed. f ALDHbright cells in MSTO-211H spheroids in the presence of DMSO or SB431542 (10 μm) were determined by ALDEFLUOR assay. All data are presented in triplicate as the mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
