Research Paper Volume 13, Issue 14 pp 18006—18017

Figure 4. Inhibition of Akt and mTOR protein kinases reversed the stimulatory effect of α2β1 knockdown on senescence of SK-Mel-147 cells. (A) Western-blotting of the cellular lysate proteins. The procedures were performed as described in Materials and Methods and the legend to Figure 1. Numbers below the bands indicate the protein band densities normalized against β-actin. Shown are representative blots. (B) Effect of PI3K/Akt inhibitor LY294002 on senescence of SK-Mel-147 cells depleted of α2β1. Cells transduced with the appropriate vectors were incubated overnight in serum-reduced medium, containing 25 μM PI3K/Akt inhibitor LY294002 followed by SA-β-Gal staining; magnification: × 200. Shown are the results of three independent experiments (M ± SEM). ρ < 0.02; I.S., insignificant. (C) Effect of mTORC1 inhibitor Rapamycin on senescence of SK-Mel-147 cells depleted of α2β1. Cells transduced with the appropriate vectors were incubated overnight in serum-reduced medium containing 200 nM Rapamycin followed by staining for β-Gal. Shown are the results of three independent experiments (M ± SEM). Vect, scramble shRNA transduced cells; α2 shRNA, α2 shRNA transduced cells; RAP, Rapamycin. *, ρ < 0.05; I.S., insignificant.