Figure 6: NIK mediates JAG1 expression in a subset of basal-like breast cancer cells.

(a) Western blotting analyses of RELA and RELB levels in nuclear extracts of control, IκBαSR- and IKKβ-expressing HCC1937 and HCC1143 cells. (b) NIK expression in HCC1937 and HCC1143 cells expressing shLuc, shNIK no. 1 or shNIK no. 2. HCC1937 and HCC1143 cells expressing shLuc, shNIK no. 1 or shNIK no. 2 were either untreated or treated with MG132 (10 μM) for 2 h. Whole-cell lysates prepared with or without MG132 treatment were subjected to western blotting. (c) Western blotting analyses of JAG1 expression in HCC1937 and HCC1143 cells expressing shLuc, shNIK no. 1 or shNIK no. 2. (d) The CSC populations (n=3, mean±s.d.; Student’s t-test) were determined by flow-cytometry analysis. (e) RELA and RELB expression and NF-κB activation in nuclear extracts of shLuc-, shNIK no. 1- or shNIK no. 2-expressing cells. Nuclear extracts were subjected to western blotting and EMSA. Whole-cell lysates were analysed as a positive control for detection of tubulin. (f) NIK expression in HCC38 cells expressing shLuc, shNIK no. 1 or shNIK no. 2. Cells expressing shLuc, shNIK no. 1 or shNIK no. 2 were either untreated or treated with MG132 (10 μM) for 2 h. Whole-cell lysates prepared with or without MG132 treatment were subjected to western blotting. (g) Western blotting analyses of JAG1 expression in HCC38 cells expressing shLuc, shNIK no. 1 or shNIK no. 2. (h) The CSC populations (n=3, mean±s.d.; Student’s t-test) were determined by flow-cytometry analysis. (i) The histone H3 acetylation profile in the NIK promoter region (n=3, mean±s.d.; Student’s t-test) was analysed using a chromatin immunoprecipitation (ChIP) assay in luminal-like (MCF7) and basal-like (HCC1937 and HCC1143) cell lines. All data are representative of three independent experiments. NS, not significant. **P<0.01 and ***P<0.001 (d,h,i).