Figure 2 : Non-cell-autonomous role of NFB in the regulation of CSC populations of basal-like cell lines.

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

Figure 2

(a) Characterization of CSC and non-CSC fractions. CSC and non-CSC fractions of MB468 and HCC1937 cells were isolated according to their CD24, CD44 and EpCAM expression levels. (b) Nuclear extracts were prepared from the sorted CSC and non-CSC populations presented in a and subjected to EMSAs. Arrows indicate the specific NF-κB/DNA complexes. (cf) NF-κB regulates the CSC population in trans. Schematic representation of the protocol for the co-culture assay (c). GFP-labelled cells were co-cultured with a 10-fold excess of cells with modified NF-κB activation. Following the indicated number of days of co-culture, the CSC populations of the GFP-labelled cells (n=3, mean±s.d.; Student’s t-test) were measured (d). After 12 days of co-culture, the GFP-labelled cells were sorted and evaluated for sphere-forming ability (n=3, mean±s.d.; Student’s t-test) (e); nuclear extracts were then prepared from the GFP-labelled and unlabelled cells for the EMSAs (f). (g) Contact between the GFP-labelled and IKKβ-expressing cells was required for the efficient expansion of the CSC population of GFP-labelled cells. GFP-labelled cells in the bottom chamber were co-cultured with control cells alone or with IKKβ-expressing cells in the same or the top chamber. After 3 days of co-culture, the CSC populations of GFP-labelled cells (n=3, mean±s.d.; Student’s t-test) in the bottom chamber were determined. (h) HCC1937 cells were untreated or treated with IL-6 (50 ng ml−1), IL-8 (100 ng ml−1) or TNFα (10 ng ml−1) for 3 days and then subjected to fluorescence-activated cell sorting (FACS) analysis and sphere-formation assay (n=3, mean±s.d.; Student’s t-test). (i) HCC1937 cells were treated with TNFα (10 ng ml−1) for 3 days in the presence of various neutralizing antibodies (2 μg ml−1) and then subjected to FACS analysis and sphere-formation assay (n=3, mean±s.d.; Student’s t-test). (j) Control or IKKβ-expressing HCC1937 cells were treated with various neutralizing antibodies (2 μg ml−1) for 3 days and subjected to FACS analysis and sphere-formation assay (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 (d,e,gj).