Knockdown of the α subunits of AMPK reverses inhibition of mTORC1, ERK and DNA synthesis induced by low doses of metformin.

<p><b>A,</b> PANC-1 cells were transfected with either non-targeting negative control (Non Target.) or 10 nM AMPKα1 and 10 nM AMPKα2 siRNA (AMPKα1, α2 siRNA) in DMEM containing 5 mM glucose and 10% FBS. After 3 days the cells were incubated either in the absence or presence of 1 mM or 3 mM metformin (as indicated) for 17 h in serum free DMEM containing 5 mM glucose. The samples were analyzed by SDS-PAGE and immunoblotting with the following phospho antibodies: S6K at Thr<sup>389</sup>, S6 at Ser<sup>240/244</sup>, ERK at Thr<sup>202</sup> and Tyr<sup>204</sup>, ACC at Ser<sup>79</sup> and Raptor at Ser<sup>792</sup> Shown here is a representative autoluminogram; similar results were obtained in 3 independent experiments. <b>B,</b> Quantification was performed using Multi Gauge V3.0. Results are expressed as the percentage of maximum (mean ±SEM; n = 3). <b>C,</b> PANC-1 cells were transfected with either non-targeting negative control (open bars) or 10 nM AMPKα1 and 10 nM AMPKα2 siRNA (black bars) in DMEM containing 3 mM glucose and 10% FBS. After 3 days the cells were incubated for 6 h in serum-free medium containing 5 mM glucose. Then, 5 nM neurotensin and 10 ng/ml insulin and metformin at either 1 mM or 3 mM were added for 17 h at 37°C prior to the addition of [<sup>3</sup>H]-thymidine for 6 h. The radioactivity incorporated into acid-insoluble pools was measured in a scintillation counter, as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114573#s2" target="_blank">Materials and Methods</a>”. Results are expressed as the percentage of maximum mean ±SEM obtained in 3 independent experiments (3 replicate cultures per point in each experiment).</p>