Research Paper Volume 14, Issue 24 pp 9877—9889
SOX2-high cancer cells exhibit an aggressive phenotype, with increases in stemness, proliferation and invasion, as well as higher metabolic activity and ATP production
Figure 1. SOX2-high MCF7 cells show an increased capacity for mammosphere formation and ATP production, with increased SOX2 protein expression levels. MCF7 cells were stably-transduced with three different stem-cell related eGFP reporter systems (SOX2-GFP, TCF/LEF1-GFP and NANOG-GFP), that measure the transcriptional activity of these nuclear factors. Then, these different cell lines were subjected to cell sorting by flow cytometry to isolate the 5% highest GFP (GFP-high) and the 5% lowest GFP (GFP-low) sub-populations. (A) The GFP-high and GFP-low sub-populations were seeded in low-attachment plates for mammosphere assays and analysed after 5 days. (B) The GFP-high and GFP-low subpopulations were plated in complete DMEM medium and incubate with the Cell-Titer-Glo 2.0 Reagent for 15 min to determinate the ATP levels. (C) SOX2 levels of GFP-high and GFP-low subpopulations were assessed by Western blotting. β-actin was used as a protein loading control. (D) SOX2 levels of GFP-high and GFP-low subpopulations were analysed by flow cytometry. Note that the use of MCF7 cells harbouring the SOX2-GFP reporter system allowed for the enrichment of a GFP-high sub-population of cells, with increased mammosphere formation, higher ATP levels, and elevated expression of endogenous SOX2, consistent with an increase in SOX2 functional transcriptional activity. Experiments were performed at least 3 times independently. Results are shown as the mean ± SD: t-test, two tailed-test, *p < 0.05, ** p < 0.01, ***p < 0.001, n.s. not statistically significant.