Figure 7: ENPP1 is a substrate of the 26S proteasome.

(a) ENPP1 expression in MCF7-luc cells stably expressing ENPP1-MF or anti-NC as a control, detected by qRT–PCR. Expression levels were normalized to those of GAPDH and data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student’s t-test. (b) A schematic illustration of the approach used in the experiments shown in c–e. (c) Immunoblot analyses of ENPP1 expression in MCF7-luc anti-NC and MCF7-luc ENPP1-MF cells treated with or without MG132 for 24 h. (d) Immunoblot analyses of ENPP1 in MCF7-luc ENPP1-MF cells grown under adherent (Ad) or mammosphere (Ma) culture conditions for the indicated times. (e) ENPP1 expression in the indicated MCF7-luc derivatives grown under adherent (Ad) or mammosphere (Ma) culture conditions. (f) Growth of the indicated MCF7-luc cell derivatives. An MTT assay was performed to determine the numbers of cells at each time point. Data are represented as the mean±s.d. of n=3 replicates. The P-values were calculated by Student’s t-test. (g) Immunofluorescent detection of ENPP1-MF and GFP in MCF7-luc ENPP1-MF cells in paraffin-embedded sections of primary tumour xenografts. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 50 μm. (h) Bioluminescent images of tumours in NOD/SCID mice injected with MCF7-luc shENPP1 cells that were treated with or without docetaxel (DOC). Alternatively, the mice were injected with MCF7-luc Zs-DR-27bs cells as a technical control. Representative images are shown for each cohort.