Figure 4 : JAG1 is crucial in the NFB-mediated stimulation of CSCs by non-CSCs.

From: NF-κB non-cell-autonomously regulates cancer stem cell populations in the basal-like breast cancer subtype

Figure 4

(a) Western blotting analyses of JAG1 expression in JAG1-overexpressing cells. (b) Flow-cytometry analysis of the CSC populations (n=3, mean±s.d.; Student’s t-test) in JAG1-overexpressing cells. (c,d) GFP-labelled cells were co-cultured with a 10-fold excess of control or JAG1-overexpressing cells. After the co-culture, the CSC populations (n=3, mean±s.d.; Student’s t-test) of the GFP-labelled cells were measured by flow-cytometry analysis (c). After 12 days of co-culture, the GFP-labelled cells were sorted and analysed for sphere-forming ability (n=3, mean±s.d.; Student’s t-test) (d). (e) HEY1 and HEY2 mRNA expression (n=3, mean±s.d.; Student’s t-test) in JAG1-overexpressing cells. (f) Western blotting of whole-cell lysates derived from control or IKKβ-overexpressing basal-like cells that additionally express either shLuc or shJAG1 (shLuc; control short hairpin RNA against luciferase gene). (g) Flow-cytometry analysis of the basal-like cells depicted in f (n=3, mean±s.d.; Student’s t-test). (h) Western blotting analysis of whole-cell lysates derived from TNFα-treated basal-like cells that express either shLuc or shJAG1. (i) Basal-like cells expressing either shLuc or shJAG1 were untreated or treated with TNFα10 ng ml−1 for 3 days, and then CSC populations (n=3, mean±s.d.; Student’s t-test) were measured by flow-cytometry analysis. (j) Western blotting analyses of JAG1 expression in shLuc- or shJAG1-expressing HCC1937 cells that were further complemented with JAG1. (k) The HCC1937 cells analysed in j were untreated or treated with TNFα (10 ng ml−1) for 3 days, and then CSC populations (n=3, mean±s.d.; Student’s t-test) were measured by flow-cytometry analysis. (l,m) Control, JAG1-expressing, IKKβ-expressing (l) and TNFα-treated (m) HCC1937 cells were untreated or treated with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (5 μM) or L685,458 (10 μM) for 3 days, and then CSC populations (n=3, mean±s.d.; Student’s t-test) were measured by flow-cytometry analysis. (n,o) Western blotting of whole-cell lysates derived from IKKβ-overexpressing (n) and TNFα-treated (o) HCC1937 cells that further express shLuc or shRBPJ. (p,q) Flow-cytometry analysis of the HCC1937 cells depicted in n and o (n=3, mean±s.d.; Student’s t-test). All data are representative of three independent experiments. NS, not significant. *P<0.05, **P<0.01 and ***P<0.001 (be,g,i,km,p,q).