Fig. 5

EGFR TKI treatment activates β-catenin signaling, which is responsible for the maintenance of DPCs. a HCC4006-Tcf/Lef-GFP reporter cells were treated with 0.1 μM erlotinib for 6 days and sorted for GFP expression by FACS into GFP^high (high β-catenin activity) and GFP^low (low β-catenin activity) cell populations, which were subsequently analyzed for Notch3, β-catenin, and stem cell markers by WB. b GFP^high and GFP^low cell populations were subjected to sphere formation assay. Quantification of total number of pulmospheres from both groups is shown. c EGFR TKI persistent pulmospheres are sensitive to β-catenin pathway inhibitors. HCC4006 cells were treated with erlotinib and TCF-GFP reporter positive cells were subjected to pulmosphere assay in the presence of DMSO or β-catenin inhibitors, XAV939, ICG-001. Quantification of total number of pulmospheres from all experimental conditions. d HCC4006-Tcf/Lef-GFP reporter cells were treated with 0.1 μM erlotinib for 6 days and cells with high and low GFP reporter activities were isolated by flow sorting. A limiting dilution assay was performed by injecting NSG mice with the number of GFP^high and GFP^low cells indicated. After 8 weeks the tumors were excised and tumor volumes and weights were measured. Solid-filled circles indicate individual tumor weights and open circles indicate no tumor grew at the site of injection. Red circles represent the group of tumors that were obtained by injection of GFP^high HCC4006 cells. Green circles represent group of tumors that were obtained by injection of GFP^low HCC4006 cells. Linear model was used to compare GFP^high and GFP^low groups within each group of cell dilution level. P values were adjusted for multiple comparisons by Holm’s procedure. For (b) and (c) error bars represent SD from biological triplicates