Figure 6.

ConA is internalized to induce STAT3–MIF–BNIP3-mediated autophagy in hepatoma cells. (a) HuH-7 cells were treated with ConA at different dose as indicated at 3 h. The lysates were subjected to detect MIF expression, phosphorylation of STAT3 (Tyr705), total STAT3, BNIP3 induction and LC3 conversion by western blotting. (b) HuH-7 cells were treated with soluble ConA (20 μg/ml) or beaded-agarose ConA (20 μg/ml) for 3 h. Cell lysates were subjected to detect phosphorylation of STAT3 (Tyr705) and total STAT3 by western blotting. (c) HuH-7 cells were treated with soluble ConA (20 μg/ml) or beaded-agarose ConA (20 μg/ml) for 24 h. Cell lysates were subjected to detect LC3 conversion by western blotting (up panel). The amount of MIF secretion in supernatant was determined by MIF-ELISA (bottom panel). (d) HuH-7 cells were treated with ConA (20 μg/ml) in the presence of either Stattic (10 μg/ml) or ISO-1 (50 μM) for 3 h. Cell lysates were subjected to detect phosphorylation of STAT3 (Tyr705), total STAT3, BNIP3 induction and LC3 conversion by western blotting. (e) HuH-7 cells were treated with ConA (20 μg/ml) in the presence or absent of Stattic (10 μg/ml) during different time periods as indicated. The MIF mRNA level was determined by RT-PCR