p21WAF1/CIP1 Expression is Differentially Regulated by Metformin and Rapamycin
Figure 6
Western blot results indicate that AMPK inhibition is associated with the reversal of metformin-induced p21 downregulation in HEK293 cells. In these experiments, the protein level of P-, cyclin D1, P-AMPKα , and P-mTO was measured in order to confirm the expected effects of treatments. mTOR and β-actin were used to control equal protein loading. (a) The cells were treated with culture medium containing 5.5 mM D-glucose. Mannitol (Man) and D-glucose (HG) were added for three days at the indicated concentrations (mM). The cells were treated with metformin (Met) for 24 h at the indicated concentrations (mM). (b) Compound C (Cc) reverses metformin-induced p21 downregulation. The cells were treated with Met and Cc for 24 h at the indicated concentrations (mM and μM, resp.). (c) HEK293 cells stably transfected with shRNA expression plasmids targeting AMPKα2 (KD) or nonsilencing (NS) were cultured with or without 8 mM metformin for 18 h in whole cell culture medium. (d) The proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal (MG132) prevents metformin-induced (Met) downregulation of p21. HEK293 cells stably transfected with shRNA expression plasmids targeting AMPKα2 (KD) or nonsilencing (NS) were cultured with or without 8 mM metformin and 10 μM MG132 overnight in whole cell culture medium. Vehicle effects were controlled by adding 0.05% DMSO to the conditions.
