Fig. 2

Adipocytes cocultivated with breast cancer cells present increased beige/brown adipocyte characteristics and undergo extensive metabolism changes. a Left, mature adipocytes in the presence or absence (NC) of MCF-7 or MDA-MB-231 tumour cells were stained with red oil. Right, the TG content in adipocytes. Breast cancer cells were transfected with miRNA-126 inhibitor, and then cocultured with mature adipocytes. The adipocytes in the CytoD group were treated with cytochalasin D (final concentration, 2 μg/ml) and 20 μg of exosomes purified from cancer-associated adipocyte conditioned medium (CA-CM). b The levels of secreted metabolites (glycerol, free fatty acids, pyruvate, lactate and β-hydroxybutyrate) enriched in media were determined by colorimetric assay. AD: adipocytes. c Glucose uptake was evaluated over time by 2-NBDG (50 μM) in glucose-free DMEM without or with insulin (INS) (50 μg/ml) or apigenin (Api) (50 μM), and the results are expressed as the mean fluorescence intensity (MFI). AD-126KD group: adipocytes cocultured with miR-126 knockdown breast cancer cells. d Raw data for the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as determined by the Seahorse XF24 analyser. The ECAR was evaluated after the addition of 10 mM glucose to adipocytes in the presence or absence of breast cancer cells. The OCR was measured in the presence of palmitate as described in the Methods. e 3T3-L1 cells were marked with LC3II-positive autophagy membranes. Autophagy membranes (LC3) visible in the GFP and RFP channels after coculture with or without MDA-MB-231 cells are shown. Green fluorescence showed that LC3-I distributed in cytoplasm. Red fluorescence showed that LC3-II distributed in autophagolysosomes. Arrowheads show LC3-II-RFP(+) only autophagosomes. f Adipocytes were cocultivated in the presence or absence of breast cancer cells. After 3 days, mRNA was extracted for qPCR analysis of the expression of the indicated genes, and (g) proteins were extracted for western blot analysis of the expression of the indicated proteins. Data are presented as the mean ± S.D. of at least three independent experiments. * P < 0.05 versus control values as blank group, # P < 0.05 versus control values as positive group