Fig. 2

Nalbulphine decreases breast cancer stem-like properties in vitro. a, b MDA-MB-231 and MCF-7 cells were treated with nalbuphine for 48 h. Levels of indicated mRNA and protein were determined by RT-PCR (a) and western blot (b) (n = 3). c MDA-MB-231 cells were treated with nalbuphine for 48 h and then subjected to in vitro limiting dilution analysis. The linear regression plot (upper) was generated by ELDA software. Stem cell frequencies are shown in the lower panel (n = 3). d Representative spheroid images derived from the Ctrl and Nal MDA-MB-231 cells (upper) (n = 3); Scale bar, 50 μm. Mammosphere sizes and number of mammospheres (bottom) (d > 50 μm) are shown. e MDA-MB-231 cells were treated with nalbuphine for 48 h, ALDH activity was then determined with DEAB-treated cells serving as negative controls (n = 3). f Mesenchymal stem cells were treated with the indicated concentration of nalbuphine for 48 h and levels of the indicated proteins were determined by western blot (n = 3). Data are presented as mean ± SEM. p-value was determined by Student’s t-test, Chi-squared test (c) and ANOVA (d) (*p < 0.05, **p < 0.01, ***p < 0.001). See also Additional file 3: Figure S2