Journal: International Journal of Oncology

Article Title: Betulinic acid inhibits stemness and EMT of pancreatic cancer cells via activation of AMPK signaling

doi: 10.3892/ijo.2018.4604

Figure Lengend Snippet: Knockdown of AMPK rescues BA-induced suppression of EMT and stemness in pancreatic cancer cells. (A) Western blotting confirmed the successful silencing of AMPK in Mia PaCa-2 and Panc-1 cells. β-actin was used as an internal loading control. (B and C) Western blot analysis suggested that silencing AMPK by siRNA reversed BA-induced inhibition of the expression of master pluripotency regulators (Sox2, Oct4 and Nanog) and EMT markers (E-cadherin and vimentin) in Mia PaCa-2 and Panc-1 cells. β-actin was used as an internal loading control. (D-F) Matrigel invasion assay revealed that silencing AMPK by siRNA reversed BA-induced suppression of the invasion of Mia PaCa-2 and Panc-1 cells. Images are representative of three independent experiments, and the invasive cells were counted and plotted. Magnification, ×200. ** P<0.01. (G-I) Tumorsphere formation assay exhibited that knocking down AMPK by siRNA reversed BA-induced inhibition of tumorsphere formation in Mia PaCa-2 and Panc-1 cells. The number of tumorspheres was counted and plotted. Magnification, ×200. * P<0.05, ** P<0.01. AMPK, 5′ adenosine monophosphate-activated protein kinase; BA, betulinic acid; EMT, epithelial-mesenchymal transition; Oct4, octamer-binding protein 4; si/siRNA, small interfering RNA; Sox2, SRY-box 2.

Article Snippet: Human epidermal growth factor (EGF) and fibroblast growth factor (FGF) were purchased from PeproTech, Inc. (Rocky Hill, NJ, USA).The antibodies used in this study were as follows: Rabbit anti-Sox2 (1:1,000 dilution; cat. no. ab97959), anti-Oct4 (1:1,000 dilution; cat. no. ab18976) and anti-Nanog (1:1,000 dilution; cat. no. ab80892) (all from Abcam, Cambridge, UK), mouse anti-β-actin (1:10,000 dilution; cat. no. 19526; KangChen Bio-Tech, Inc., Shanghai, China), mouse anti-E-cadherin (1:1,000 dilution; cat. no. sc-71008), rabbit anti-cluster of differentiation (CD)133 (1:1,000 dilution; cat. no. sc-11406), mouse anti-aldehyde dehydrogenase (ALDH)1 (1:1,000 dilution; cat. no. sc-374149) and mouse anti-epithelial cell adhesion molecule (EpCAM) (1:1,000 dilution; cat. no. sc-66020) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and rabbit anti-vimentin (1:800 dilution; cat. no. 12826), anti-AMPK (1:800 dilution; cat. no. 5831) and anti-phosphorylated (P)-AMPK (Thr172) (1:800 dilution; cat. no. 2535) (all from Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Western Blot, Inhibition, Expressing, Invasion Assay, Tube Formation Assay, Binding Assay, Small Interfering RNA