Cell Culture, Calcium Depletion, and AMPK Signaling

HAECs and HUVECs were grown in MV2 medium (Promocell, Heidelberg, Germany) and passaged when at 80% confluence. Cells were used for experiments between passages 3 and 6 as described previously [33 (link)–35 (link)]. For experiments in which extracellular Ca²⁺ was depleted, cells were incubated in KRH buffer [20 mmol/l HEPES–NaOH (pH 7.4), 119 mmol/l NaCl, 5 mmol/l NaHCO₃, 5 mmol/l glucose, 4.8 mmol/l KCl, 1.2 mmol/l MgSO₄, 1.2 mmol/l NaH₂PO₄, 2.5 mmol/l CaCl₂] or KRH without CaCl₂ supplemented with 1 mmol/l EGTA for 2 h prior to stimulation with VEGF or AICAR. HeLa cells and HEK293 cells were cultured in DMEM supplemented with 10% (v/v) foetal calf serum (FCS). HeLa cells stably expressing LKB1 and MEFs lacking AMPKα1 and AMPKα2 (AMPK KO MEFs) were cultured as described previously [32 (link),35 (link)].