Figure 6: Functional analysis of ENPP1 in MCF7-luc cells.
From: Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1
(a) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. (b) Quantification of the SP fractions shown in a, determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n=3 replicates. (c) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. (d) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. (e) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC50 value. Data are represented as the mean±s.d. of n=3 replicates. (f) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. (g) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.
