Fig. 2

Critical roles of lipid metabolic remodeling in breast cancer cell death induction by globular adiponectin. A-B MCF-7 (A) and MDA-MB-231 cells (B) were treated with gAcrp (1 μg/mL) or TVB-3166 (200 nM), a pharmacological inhibitor of FASN, for 48 h. Lipid raft microdomains were labeled with Alexa fluor 488-conjugated Cholera toxin B (CT-B) as described in Methods (green color). Representative images from three independent experiments were presented (upper panel) along with quantification of the corrected total cell fluorescence (CTCF) for CT-B (lower panel), in which CTCF was determined by integrated density - (area of selected cell × mean fluorescence of background readings) as previously described [38]. C-D MDA-MB-231 cells were treated with gAcrp for 48 h, followed by further stimulated with Wnt3a (100 ng/mL) for 6 h. C Lipid raft (green) was stained with Alexa fluor 488-conjugated CT-B and membrane p-LRP6 was labeled with a specific primary antibody and an Alexa fluor 594-conjugated secondary antibody (red). Scale bar: 20 μm. D p-LRP6, LRP6, and non-phospho- (active) β-catenin levels were examined by western blot analysis. Bar diagram shows quantification of blots. E-I MCF-7 (E and G) and MDA-MB-231 (F, H, and I) cells were treated with gAcrp (1 μg/mL) or TVB-3166 (200 nM) for 48 h in serum-free culture media in the absence of presence of BSA-conjugated palmitic acid (50 mM). Cell viability (E-F) and capase-3/7 activity (G-H) were determined at the end of treatment period as described in Methods. I Cells were double-stained with FITC-annexin V and 7-AAD, and subjected to flow cytometry analysis. Bar diagram shows the percentage of annexin V-positive cells that represents apoptotic cell population. * indicates p < 0.05 compared to control cells; # indicates p < 0.05 compared to cells treated with gAcrp alone; $ indicates p < 0.05 compared to cells treated with TVB-3166 alone; n=3 except where specifically indicated in Figures