Figure 5

Figure 5

GW296115X activates AMPK resulting in autophagosome formation and elimination of intracellular MRSA. (A) Chemical structure of GW296115X. (B, C) GW296115X was tested at different concentrations to determine its antimicrobial potency (B) and level of cytoxicity (C) in MRSA-infected HeLa cells. (D) The KinMap kinase phylogenetic tree shows the kinase inhibition profile of GW296115X as determined by Caliper assay (Elkins et al., 2016). Each circle represents a kinase target that is inhibited > 50% at 0.1 µM (purple) or 1 µM (blue) concentration. (E) Phosphorylation of ACC1 at Ser80, a direct target of AMPK, was determined by flow cytometry and expressed as fold change in geometric mean fluorescent intensity (gMFI) compared to untreated HeLa cells. The AMPK-activating compound 991 was used as a positive control. (F) Phosphorylation of AMPK at Thr172 was determined by western blot and expressed as the ratio between phosphorylated and unphosphorylated AMPK. (G) Autophagosome formation was determined by western blot and expressed as the ratio between lipidated, membrane-bound LC3 (LC3-II) and cytosolic LC3 (LC3-I). Abbreviations: BAF = bafilomycin A1; RAP = rapamycin. The combination of lysosome inhibitor bafilomycin A1 and mTOR -inhibitor rapamycin results was used as a positive control that results in LC3-II accumulation. (H, I) The interaction between bafilomycin A1 and GW296115X and their effect against intracellular MRSA (H) and host cell viability (I) were determined. (J, K) The effect of two other autophagy-inducing compounds 991 and rapamycin against intracellular MRSA (J) and host cell viability (K) were determined. Each datapoint represents the results of an independent experiment. Statistical significance of observed differences was tested in comparison to the DMSO controls (*p < 0.05; **p < 0.01; ***p < 0.001).