Fig. 2: Effect of fisetin on AMPK signaling pathway.

To promote autophagosome generation, cells were treated with 20 μM Chloroquine (CQ) for 2 h before collection. a Western blotting analyses of LC3B in control (Con) and fisetin treated PANC-1 cells. Cells were treated with fisetin (200 μM) for 24 h and 48 h. Quantitative data of optical band densitometry are shown. Data are presented as mean ± SD (n = 5), *P ≤ 0.05, ****P ≤ 0.0001. b Analyses of autophagy by observation of the immunofluorescence of LC3B (green)in PANC-1 cells. Cells were treated with fisetin (200 μM) for 48 h. And CQ were used as described previously. The nucleus were counter stained with DAPI (blue). Scale bars 10 μm. Comparative changes in the LC3B spots number in control and treatment groups. The spots were counted by manual scoring. Results are mean ± SD, **P ≤ 0.01. c Fisetin-mediated changes in AMPK (p-AMPK) and mTOR target protein p70S6K (p-p70S6K) in PANC-1 cells. Effect of fisetin on activation of AMPK resulted in inhibition of mTOR target protein. Data are presented as mean ± SD(n = 3), *P ≤ 0.05,**P ≤ 0.01. d Western blotting analyses of AMPK (p-AMPK). Cells were treatment of AMPK inhibitor compound C (CC) (20 μM)for 4 h before collection. Fisetin and CQ were used as described previously. The level of p-AMPK was reduced by CC. e Western blotting analyses of LC3B. Fisetin, CC, and CQ were used as described previously