Fig. 5

Dual reporter cell lines identify an ER and NFĸB-driven stem-like cell population. A, B ERE-mCherry and NFĸB-RE-eGFP activity in 2D-cultured MCF-7 dual reporter cells treated with E2 (10 nM), hTNFα (10 ng/ml), ICI (1 µM) and/or IKK7 (1 µM) for 24 h was measured using a Celigo imaging cytometer. Bar charts represent the percentage of mCherry confluence (A) and eGFP (B) confluence normalized to total brightfield confluence for each treatment group. C ERE-mCherry and NFĸB-RE-eGFP activity in MCF-7 MS was measured over time. D A representative image of MS derived from MCF-7 dual reported cell line is shown (bar = 100 µm). E A schematic of sorting experiments performed on MS derived from the MCF-7 dual reporter cells is presented. F MS derived from MCF-7 dual reporter cells were collected, trypsinized and sorted for 4 cell populations, based on expression of fluorescent proteins eGFP and mCherry. A secondary MS assay was performed on 4 sorted populations and secondary MFE for each group is plotted. G Cell distribution of secondary MS is plotted for each group based on ERE-mCherry and NFĸB-RE-eGFP activity. H The expression of DEGs of MS Cluster was determined in each cell population by QPCR. Fold change, normalized to dual-negative cell population, for each gene is presented on a heatmap. K, L Distribution of z-scores for the Hallmark ER (I) and NFĸB (J) signatures are shown on a per cell basis. K Each cell from MS Cluster 1 was assigned to one of the groups based on z-scores for ER and NFĸB signatures: dual-negative cells (white), dual-positive cells (yellow), ER-active cells (red), NFĸB-active cells (green). The percent change in abundance of MS Cluster 1 relative to the total population for each group is shown. L A bar charts representing GFD15 mRNA expression in each sorted group in (K). M, P The role of GDF15 on secondary MS formation was examined using an anti-GDF-15 antibody (200 ng/ml) on 2 sorted cell population from: dual-positive (M) and ER-active (N). *P < 0.05, ***P < 0.001, ns not significant