Figure 8

hGX-induced LD accumulation is associated with activation of AMPKα. (A) MDA-MB-231 cells were treated with OA (100 μM) and hGX (1 nM) in complete culture medium for 48 h in the presence of etomoxir (Eto; 20 μM), triacsin C (TrC; 2 μM), AICAR (500 μM) or bezafibrate (Bez; 500 μM). Free OA was incubated in complete culture medium for 1 h before addition to the cells. Cell lysates were analyzed for the presence of Thr172-phosphorylated AMPKα (p-AMPKα), total AMPKα and β-actin loading control by immunoblotting and densitometry. The amounts of p-AMPKα obtained from three separate experiments were normalized to total AMPKα protein levels and quantified relative to untreated controls (B). (C) Cells were treated as in (A). Prolonged incubation (48 h) of proliferating MDA-MB-231 cells with AICAR (500 μM) abolished the hGX-induced (1 nM) LD formation. Cellular LD content was determined by Nile red staining. (D, E) Serum-starved MDA-MB-231 cells were treated with hGX (10 nM) in serum-free medium containing 0.02% FAF BSA for 96 h in the presence or absence of AICAR (500 μM). After 96 h, cellular LD content was determined by Nile red staining, showing that AICAR prevented hGX-induced LD formation (D). Cell survival was assessed with the TMRM/YO-PRO-1 apoptosis assay, indicating that AICAR alone has a pro-survival effect in MDA-MB-231 cells, thus effectively masking the positive effect exerted by hGX. Values on the graphs are means ± SD of at least two experiments performed in duplicate and results that are statistically significant over control samples are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA with Bonferroni adjustment).