Figure 2

AMP-activated protein kinase (AMPK) inhibits apoptosis through phosphoprotein enriched in astrocytes 15 kDa (PEA15) phosphorylation. Primary human mammary epithelial cells (HMECs) cultured under following conditions were harvested and subjected to immunoblot analysis for the specified proteins: (A) HMECs seeded in adherent (ADH) condition for six days or in suspension (in ultra-low (UL) plates) for two, four or six days; n = 3. (B) HMECs seeded in suspension in the presence of 100 μM AMPK activator (A769662), 10 μM AMPK inhibitor (Compound C) or dimethyl sulphoxide (DMSO) (vehicle control) for a week; n = 3. (C) HMECs seeded in UL plates and transfected with control small interfering RNA (siRNA) or siRNA targeting AMPK α2 (Dharmacon) were harvested after one week; n = 4. (D) HMECs seeded in ADH condition or in suspension in UL plates for a week. Graph represents densitometric analysis of western blots to quantify phospho PEA15 Ser¹¹⁶ relative to total PEA15. Error bars represent standard error of the mean (SEM); (n = 6). (E) HMECs seeded in UL plates for 24 hrs and treated with DMSO (vehicle control), 100 μM AMPK activator (A769662), or 10 μM AMPK inhibitor (Compound C) for a period of 2 hrs; n = 3. (F) HMECs seeded in UL plates and treated with increasing amounts of AMPK inhibitor Compound C, in the presence of a constant amount of AMPK activator A769662, for 2 hrs; n = 3. (G) HMECs seeded in UL plates and transfected with control siRNA or siRNA targeting AMPK α2 and harvested two days following transfection; n = 4. (H) HMECs seeded in UL plates and transfected with control siRNA or siRNA targeting AMPK α2 were treated with 100 μM AMPK activator (A769662) after two days following transfection for 2 hrs and harvested, n = 3.