Resveratrol Prevents Oxidative Stress-Induced Senescence and Proliferative Dysfunction by Activating the AMPK-FOXO3 Cascade in Cultured Primary Human Keratinocytes
Fig 6
Effects of AMPK knockdown, AMPK inhibitor Compound C, SIRT1 inhibitor Ex527, and SIRT1 knockdown on 3H -thymidine incorporation.
(a) Keratinocytes were infected with lentivirus vector expressing non-targeting control shRNA (shNegative) or AMPK alpha1 (shAMPKa1). Cells were then treated with 20 µM H2O2, 1 nM insulin and resveratrol (Res) as described in Fig. 5. Knockdown of AMPK abrogated the effects of resveratrol (which are shown in Fig. 5c). (b) Non-infected cells were treated with H2O2 and resveratrol as in 6a, but incubated with 1 µg/ml Compound C (CC), an AMPK inhibitor, 30 min. prior to the addition of resveratrol. Compound C inhibited the effects of resveratrol on 3H -thymidine incorporation. (c) Keratinocytes were infected with lentivirus vector expressing non-targeting control shRNA (shNegative) or SIRT1 (shSIRT1) that reduced total SIRT1 by about 70%. Knocking down SIRT1 had no effect on resveratrol-induced changes in 3H -thymidine incorporation. (d) Non-infected cells were incubated with H2O2, insulin and resveratrol as in 6a, and 10 µM Ex527 was added 10 min. prior to the addition of resveratrol. Ex527 treatment did not alter 3H -thymidine incorporation suggesting that inhibition of SIRT1 did not modulate the effect of resveratrol. For (a) and (c), numbers inside parenthesis denote n.