Fig. 4

Activation of lipophagy by globular adiponectin in breast cancer cells. A-B MCF-7 (A) and MDA-MB-231 (B) cells were treated with 1 μg/mL of gAcrp for different time periods. The expression levels of autophagy-related genes, including p62, Beclin-1, Atg5, and LC3I/II were examined by western blot analysis. C-F Cells were cultured in the media containing 10 μM of oleic acid/BSA to induce lipid droplet formation and treated with 1 μg/mL of gAcrp for 6 h. C-D Lipid droplets and LC3 were detected by labelling with Nile red (red), and incubation with an anti-LC3 primary antibody followed by Alexa fluor 488-conjugated antibody (green), respectively. E-F Lipid droplets and lysosomes were labeled with Bodipy 493/503 (green) and Lysotracker (red). Co-localization rate was determined by Mander’s overlap coefficient using Image J software. Scale bar: 20 μm. G-H Cells were treated with gAcrp for the indicated time duration. Free fatty acid levels were measured at the end of treatment periods as indicated in the methods. I MDA-MB-231 cells were treated with gAcrp (1 μg/mL) for 8 h. Fatty acid oxidation (FAO) was determined as described in the methods. * denotes p < 0.05 compared to control cells; n=3 except where specifically indicated in Figures