Fig. 6

TGF-β/Akt/Smad signaling pathway in HCC spheres is activated by RIG-I KD. a RIG-I knocked-down and reference SMMC-7721 tumorspheres were stimulated with 2 ng/ml of TGF-β1 for 1 h. Western blot analysis was performed using the indicated antibodies. b RIG-I knocked-down and reference SMMC-7721 cells were transfected with Akt1/2 siRNA. Akt-deficient tumorspheres were stimulated with TGF-β1 in the same manner as (a), and Western blot analysis was performed using the indicated antibodies. c RIG-I knocked-down and reference SMMC-7721 tumorspheres were stimulated with TGF-β1 in the same manner as (a). Smad2/p-Smad2 and Akt/p-Akt interaction was detected by immunoprecipitation with anti-Smad2 antibody (upper panel) or anti-Akt antibody (lower panel), IgG being used as a negative control antibody. Levels of GAPDH protein were monitored as a loading control. The values are presented as means ± SD (n = 3). **P < 0.01