Figure 4

AMP-activated protein kinase (AMPK)-phosphoprotein enriched in astrocytes 15 kDa (PEA15) axis is critical for the anchorage-independent growth of breast cancer cells. (A) Michigan Cancer Foundation 7 breast cancer cell line (MCF7), BT-474, and primary breast cancer-derived cells cultured in adherent (ADH) condition or as cancer spheres (CS) for a week were harvested and subjected to immunoblotting; n = 3. (B) MCF7 cells cultured in methylcellulose for a week in the presence of 10 μM AMPK inhibitor Compound C or dimethyl sulphoxide (DMSO) (vehicle control) were subjected to immunoblotting. Graph represents number of spheres formed/20 fields. Error bars represent standard error of the mean (SEM); n = 3. (C-F) Adherent MCF7 breast cancer cells were transfected with specified small interfering RNA (siRNA) oligos/plasmids (see below). Two days posttransfection, 1 × 10⁵ cells/35 mm dish were seeded in methylcellulose. After 48 hrs, cells were retrieved from some dishes and subjected to immunoblot analyses for the specified proteins. Parallel dishes were allowed to form spheres; graph represents sphere formation at the end of a week, error bar represents SEM: (C) control siRNA or siRNA targeting AMPK α2, n = 4; (D) control siRNA or siRNA targeting PEA15, (n = 4); (E) control siRNA or siRNA targeting PEA15 and seeded in methylcellulose in the presence of 100 μM AMPK activator, A769662; DMSO served as vehicle control, n = 4; (F) transfected with CSCG constructs expressing flag-tagged wild-type (WT) or S116A mutant of PEA15, n = 4. (G) MDAMB231 cells were treated with DMSO or 100 μM AMPK activator, A769662, and immunoprecipitated with anti-Fas-associated death domain protein (FADD) antibody. The immunoprecipates were resolved by SDS-PAGE followed by immunoblotting for specified proteins, n = 3. (H) BT 474 cells stably expressing flag-tagged WT-PEA15 or PEA15-S116A mutant were seeded in methyl cellulose for two days, retrieved and immunoprecipitated with anti-flag antibody. The immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting for specified proteins, n = 3.