Figure 4. Effects of APG and TRAIL on NF-κB, PI3K/AKT and JNK signaling pathway.

(a) A549 cells were treated with APG at different concentrations (0, 10, 20, 40 μM) in the absence or the presence of TRAIL (25 ng/mL) for 24 h. Nuclear proteins were extracted and subjected to Western blotting for p65 detection. Lamin B was used as loading control. Additionally, the whole cell extracts with the same treatment were prepared and analyzed for IκB-α and p-IκB-α expression. (b) A549 and H1299 cells were treated with APG at different concentrations (0, 20, 40 μM) in the absence or the presence of TRAIL (25 ng/mL) for 24 h. AKT, p-AKT, PI3K, and p-PI3K proteins in whole cell lysates were determined with specific antibodies. GAPDH was used as loading control. (c) A549 and H1299 cells were treated with APG (20 μM), TRAIL (25 ng/mL), LY294002 (5 μM), or their combination for 24 h before determination of cell death by flow cytometry analysis. The experiments were carried out independently in triplicate; representative data are shown. Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***P < 0.001. (d) A549 cells were treated with APG at different concentrations (0, 20, 40 μM) in the absence or the presence of TRAIL (25 ng/mL) for 24 h. c-JUN, p-c-JUN and p-JNK protein in whole cell lysates were determined with specific antibodies. GAPDH was used as loading control. For (a,b,d), all gels run under the same experimental conditions and the representative images of three different experiments were cropped and shown. Densitometric quantification of the immunoblot data is also shown and data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.