Figure 2.

Cellular efficacy of STO-609 and SGC-CAMKK2-1 in inhibiting Ca²⁺/CaMKK2-dependent phosphorylation of AMPK. (A) LKB1-deficient wild-type (WT) or CaMKK2 knock-out (KO) A549 cells were treated with either vehicle (0.1% DMSO), ionomycin (1 μM) or AICAR (2 mM) and cell extracts were subjected to immunoblot analysis with the indicated antibodies. Cell extracts from mouse embryonic fibroblasts (MEFs) were used as control for LKB1. Representative immunoblots from two independent experiments are shown. An arrow indicates the band of interest. (B) WT A549 cells were serum starved for 2 h and pre-incubated for 30 min with vehicle (−) or increasing doses of STO-609, SGC-CAMKK2-1 or SGC-CAMKK2-1N followed by treatment with or without ionomycin (1 μM) for 5 min. Cell extracts were subjected to immunoblot analysis with the indicated antibodies. Representative immunoblots from three independent experiments are shown. (C) Percent (%) changes of AMPKα Thr172 phosphorylation levels relative to the ionomycin-treated condition (100%, 0 inhibitor) shown in (B) with data points represented as mean ± SEM. n = 2 per treatment condition.