Figure 3.

AMPK inhibitor, compound C, stunts Akt-dependent signaling, and PC3 cell survival. (A) Cell viability was evaluated by WST-1 dye conversion at 24 and 48 hours. PC3 cell viability graph bars in response to compound C (20 µM) are shown for control and CCL2-stimulated cells. The data are representative of n = 5 assays for each CCL2-stimulated and control cells. SD bars are depicted for each data point. (B) Western blot depicting reduced AMPK activity in response to compound C (20 µM) in cells treated or not with CCL2 as evaluated by raptor phosphorylation (Ser⁷⁹²). The blots also show how compound C hinders Akt activation by inhibiting phosphorylation of key activation sites (Ser⁴⁷³ and Thr³⁰⁸) and thus preventing phosphorylation of a downstream target PRAS40 (Thr²⁴⁶). (C) Immunoblot analysis illustrating the effect of compound C at 24 hours on p70^S6K phosphorylation (Thr³⁸⁹), survivin expression, and the autophagic marker LC3-II. (D) CCL2 regulates AMPK signaling through an Akt-independent mechanism. Serum-starved, CCL2-stimulated PC3 cells were treated with increasing concentrations (1.25, 2.5, and 5 µM) of Akt inhibitor Akti-X (Calbiochem). Western blot analysis reveals how Akti-X effectively inhibits Akt activation/phosphorylation (Ser⁴⁷³ and Thr³⁰⁸) resulting in a reduced PRAS40 phosphorylation (Thr²⁴⁶). AMPK phosphorylation (Thr¹⁷²) and the induction of autophagy (LC3-II) were evaluated in response to Akti-X. Phospho-specific expression was verified by comparison with total respective proteins, and β-actin is included as a loading control.