Figure 3

Linifanib activates autophagic flux in hepatocarcinoma cells.

(A). Immunoblotting for Beclin-1, ATG5, ATG7 and p62 using lysates from cells treated with linifanib for 24 h. (B). Immunoblotting for LC3 in Bel-7404 cells treated with 2.5 μM linifanib in the presence or absence of 5 μM CQ or 5 mM 3-MA. Relative quantity of LC3-II was calculated by ImageJ densitometric analysis and normalized by GAPDH. (C). Quantification of green or yellow puncta-positive cells by using a threshold of >5 dots/cell after treatment with 2.5 μM linifanib in the presence or absence of autophagy inhibitors for 24 h. (D). Bel-7404 RLuc–LC3^WT and RLuc–LC3^G120A cells were treated with linifanib or 125 nM rapamycin. Luciferase activity was measured at 24 and 48 hours after treatment. Results shown are the mean ± SD of at least three independent experiments. *P < 0.05. ** P < 0.01.