Fig. 3

Expression of ADM in MCF7 cells and ADM receptors in adipocytes. a-b, Expression of ADM and CA IX in MCF7 cells cultured in different conditions. Expression of ADM and CA IX was assessed by real-time RT-PCR and normalized for the expression of 36B4 mRNA. Expression was measured in cells grown as 2D monolayers in the presence of 21 or 2% O2 for 48 h or as mammospheres for 7 days. The means ± SEM were calculated from three independent experiments, with determinations performed in duplicate (*p < 0.05, ** p < 0.01). c, Expression of ADM in mammospheres. MCF7 cells were grown as mammospheres for 7 days. Expressions of ADM (green) and PLIN1 (red, to label adipocytes) are shown. DAPI was used to label the nuclei (Blue). The fluorescence recorded for each channel is shown in separated images (see Supp. Figure 3). Note that ADM was not expressed in adipocytes, but in mammospheres. Scale bar: 50 μm. d-e ADM receptors expression in hMADS (d) and breast-adipocytes (e). Time course for the expression of CRLR and RAMP2, was assessed by real-time RT-PCR and normalized to the expression of 36B4 mRNA. Expressions were measured in cells that received (red bars) or did not receive (blue bars) the differentiation cocktail for the indicated number of days. The means ± SEM were calculated from three independent experiments, with determinations performed in duplicate (*p < 0.05, ** p < 0.01)