Fig. 5

Roles of SIRT-1 in fatty acid metabolic reprogramming by globular adiponectin in breast cancer cells. A-B MCF-7 (A) and MDA-MB-231 (B) cells were treated with gAcrp as indicated. SIRT-1 expression was determined by western blot analysis. C MCF-7 cells were pretreated with EX527 (5 μM) for 2 h, followed by incubation with gAcrp (1 μg/mL) for further 8 h. The protein levels of SREBP-1 and FASN were examined by western blot analysis. D MCF-7 cells were transfected with a siRNA targeting SIRT-1 or a scramble control siRNA for 36 h, followed by treatment with gAcrp (1 μg/mL) for 8 h. (Upper panel) The gene silencing efficiency was monitored by western blot analysis. (Lower panel) The expression levels of FASN and SREBP-1 in SIRT-1 knockdown cells were examined after gAcrp treatment. E MCF-7 cells were pretreated with EX527 for 2 h, followed by further incubation with gAcrp (1 μg/mL) for 12 h. mRNA levels of FASN, ACC-1, ACLY, and FADS2 were measured by RT-qPCR. F-H MCF-7 cells were transfected with SIRT-1 siRNA (25 nM) for 36 h, followed by treatment with 1 μg/mL of gAcrp for 24 h (F), 48 h (G), or 6 h (H). F Cellular neutral lipid content was determined by Bodipy 493/503 uptake assay. G Cells were labeled with Alexa fluor 488-conjugated CT-B and lipid raft microdomains were observed under a confocal microscope. H Lipid droplets were stained with Nile red and autophagosomes were labeled with an Alexa fluor 488-conjugated anti-LC3 antibody. The overlapping between lipid droplets (red) and autophagosomes (green) were observed under a confocal microscope. The Mander’s overlap coefficient was used to test the colocalization of lipid droplets and autophagosomes. Scale bar: 20 μm. I-M MCF-7 cells were transfected with SIRT-1 siRNA (25 nM) (I) or pretreated with EX527 for 2 h (J-M), followed by further treatment with 1 μg/mL of gAcrp for 48 h. I and J Cell viability was measured by MTS assay. K-L The apoptosis level was determined using caspase-3/7 activity (K) and TUNEL assay (L) as indicated in the methods. M Expression levels of Bax and Bcl2 were examined by western blot analysis. * denotes p < 0.05 compared to control; # denotes p < 0.05 compared to cells treated with gAcrp alone; n=3 except where specifically indicated in Figures