Figure 5. Knockdown of the α subunits of AMPK reverses inhibition of mTORC1, ERK and DNA synthesis induced by low but not high doses of berberine.

A, 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 berberine for 17 h in serum free DMEM containing 5 mM glucose. Then, the cells were stimulated for 1 h with 5 nM neurotensin and 10 ng/ml insulin and lysed with 2X SDS-PAGE sample buffer. The samples were analyzed by SDS-PAGE and immunoblotting with the following phospho antibodies: S6K at Thr³⁸⁹, S6 at Ser^240/244, and ERK at Thr²⁰² and Tyr²⁰⁴, ACC at Ser⁷⁹ and Raptor at Ser⁷⁹². Shown here is a representative autoluminogram; similar results were obtained in 4 independent experiments. B, Quantification was performed using Multi Gauge V3.0. Results are expressed as the percentage of the maximum (mean ±SEM: n = 4). C, 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 5 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 the indicated concentration of berberine were added for 17 h at 37°C prior to the addition of [³H]-thymidine for 6 h. The radioactivity incorporated into acid-insoluble pools was measured in a scintillation counter, as described in “Materials and Methods”. Results are expressed as the percentage of maximum mean ± SEM obtained in 4 independent experiments (3 replicate cultures per point in each experiment).