Fig. 5

Reactivation of WNT pathway supports MYC-induced stem cell features. a qRT-PCR of WNT pathway-related genes on IMEC WT, IMEC-MYC, and M2 clones #1 and #2, normalized on spike-in RNAs. Data are means ± SEM (n = 3) (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test). b FACS analysis showing the GFP signal of IMEC-MYC-7TGP cultured in adhesion or as mammospheres. c On the left, phase contrast images showing IMEC-MYC-7TGP cultured in low adhesion conditions. Scale bar, 100 µm. On the right, FACS analysis showing GFP signal and dye retention profile of IMEC-MYC-7TGP cultured in low adhesion conditions. d Scheme representing GFP^high and GFP^low cells sorting from IMEC-MYC-7TGP, which gave rise to GFP^high- and GFP^low-derived 1° Spheres (M1). GFP^high-derived 1° Spheres underwent a second single-cell sorting of GFP^high and GFP^low cells, which gave rise to GFP^high- and GFP^low-derived 2° Spheres (M2). Representative FACS analysis showing GFP signal and median fluorescence intensity (MFI) of sorted IMEC-MYC-7TGP and GFP^high-derived M1 clones are reported. e On the left, single-cell spheres formation efficiency (SFE) of GFP^high and GFP^low cells sorted from IMEC-MYC-7TGP, which gave rise to M1 clones. MFI of GFP^high- and GFP^low-derived M1 clones is reported. On the right, single-cell SFE of GFP^high and GFP^low cells sorted from GFP^high-derived M1, which gave rise to M2 clones. MFI of GFP^high- and GFP^low-derived M2 clones is reported. Data are means ± SEM (n = 4). f GSEA of mammary stem cells (MaSCs) gene signature in freshly sorted GFP^high and GFP^low cells (n = 3). g GSEA of lung, bone, and brain metastatic signatures in freshly sorted GFP^high and GFP^low cells (n = 3)