Modulation of mitochondrial respiration activity in breast cancer cells: mitochondrial respiration is attenuated after inactivation of p70S6K either by rapamycin or p70S6K shRNA and activated after over expression of p70S6K. (A) Effect of rapamycin on mitochondrial respiration. Breast cancer cells (1x10^6 cells) were treated with 0.1 μM rapamycin for 24 hours, and mitochondrial respiration was measured using the oxytherm system (Clark electrode) in growth medium. (B) Inhibition of p70S6K phosphorylation by rapamycin. BT474 cells were treated with 0.1 μM rapamycin for up to 24 hours and cell lysate were analyzed for phospho-p70S6K by western blotting. (C) Effect of AMP activator (AICAR) on the mitochondrial respiration of breast cancer cells. Top panel, activation of AMP-activated protein kinase (AMPK) by AICAR. Cells were treated with 1 mM AICAR for 24 hours and cell lysates were probed for AMPK status. Bottom panel, effect of AICAR on mitochondrial respiration. Cells (1 × 10^6 cells) were treated with 1 mM AICAR for 24 hours and mitochondrial respiration was evaluated with the Clark electrode in growth medium. (D) Effect of over expression of p70S6K on mitochondrial respiration in MDA468 cells. Left panel, over-expression of P70S6K was first confirmed by western blot. Right panel, mitochondrial respiration of MDA468-p70S6K, vector control, and the parental cell line MDA468 (1 × 10^6 cells) were determined using oxytherm system. (E) Knockdown of p70S6K in BT474 decreased the mitochondrial respiration. Top panel, knockdown of p70S6K in BT474 was first confirmed by western blot in several clones. BT474 cells were transfected with control ShRNA (FF3 clones) and p70S6K ShRNA (clones D7 and B8), then clones were selected by controlling the expression of p70S6K. Bottom panel, comparison of the mitochondrial respiration between clones of ShRNA (FF3, B8 and D7) and the parental cell line BT474 (1 × 10^6 cells) measured by Clark electrode. Each bar graph represents mean ± SD from three experiments; *Pp <0.05. One representative from at least three independent experiments is shown for each western blot.