Figure 1

Mammospheres show elevated AMP-activated protein kinase (AMPK) activity. (A) Phase-contrast microscopic images (left) of freshly isolated primary human mammary epithelial cells (HMECs) cultured for a week as monolayers in adherent (ADH) condition in regular tissue culture (TC) dishes, or as mammospheres (MS) in suspension in ultra-low attachment plates (UL plates); magnification ×200. Experimental strategies (right) involving HMECs seeded in suspension (also see Methods). (B and C) Primary HMECs cultured in ADH condition or in suspension as MS were harvested at the end of a week and subjected to immunoblot analyses for the specified proteins. Graphs represent densitometry analyses of western blots to quantify phosphoprotein relative to total protein. Error bars represent standard error of the mean (SEM), (n = 6). The phosphorylated (p)AMPK antibody recognizes both AMPK α1 and α2 isoforms; antibodies against total AMPK recognize both α1/2 together, or only individual isoforms α1 or α2; α-tubulin served as loading control for all western blot experiments. (D) Primary HMECs were cultured in UL plates in the presence of 10 μM AMPK inhibitor, Compound C (CC); dimethyl sulfoxide (DMSO) served as vehicle control. Two days after treatment, a quarter of the cells were harvested and subjected to immunoblot analyses for specified proteins, while the rest were scored for total number of MS formed at the end of a week (graph). Error bar represents SEM, (n = 4). (E). Primary HMECs seeded in UL plates were transfected with control small interfering RNA (siRNA) or siRNA targeting AMPK α2 (Dharmacon, Thermo Fisher Scientific, Waltham, MA, USA). Two days posttransfection, a quarter of the cells were harvested and subjected to immunoblot analyses for the specified proteins, while the rest were scored for total number of MS formed at the end of a week (graph). Error bars represent SEM; n = 4.