Figure 2.

Wogonin activates the Beclin-1/PI3K signaling pathway. (A and B) HPCCs were treated with 40 µm wogonin or 0.1% DMSO for 12 h. Antibodies targeting (A) Beclin-1 or (B) PI3K were added to immunoprecipitate the Beclin-1- or PI3K-containing complexes and then IB for PI3K or Beclin-1 was performed. The results revealed that Beclin-1 and PI3K were pulled down after PI3K and Beclin-1 were downregulated, respectively, and wogonin could upregulate Beclin-1 and PI3K, which indicated that Beclin-1 and PI3K were bound to one another, and wogonin could promote integration to form the Beclin-1/PI3K complex. (C) HPCCs were transfected with control siRNA or siRNA-Beclin-1; at 36 h post-transfection, HPCCs were treated with 0.1% DMSO or 40 µM wogonin for 24 h and then immunoblotted for LC3 and Beclin-1, revealing that LC3 expression was decreased following Beclin-1 knowckdown. (D) HPCCs expressing LC3-GFP were transfected with control siRNA or siRNA-Beclin-1. At 36 h post-transfection, HPCCs were treated with 0.1% DMSO or 40 µM wogonin for 24 h, and the LC3-GFP puncta-positive cells were counted (≥5 puncta was considered positive). After Beclin-1 was knocked down, the number of positive cells was significantly decreased, but not reduced to zero, which indicated that the Beclin-1/PI3K complex was involved in, but not solely responsible for, wogonin-induced autophagy. Data are presented as the mean ± standard deviation from triplicated experiments. **P<0.01; ***P<0.005. HPCC, human pancreatic cancer cell; CQ, chloroquine, LC3, microtubule-associated protein 1A/1B-light chain 3; GFP, green fluorescent protein; TEM, transmission electron microscopy; DMSO, dimethyl sulfoxide; siRNA, small interfering RNA; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Con, control; IgG, immunoglobulin G; IB, immunoblotting; IP, immunoprecipitation.