Research Paper Volume 14, Issue 24 pp 9877—9889

Figure 7. SOX2-high MDA-MB-231 cells form mammospheres with greater efficiency, produce more ATP, and show a loss of E-cadherin expression. MDA-MB-231 cells stably-transduced with the SOX2-GFP construct were subjected to FACS sorting to isolate the 5% highest GFP (GFP-high) and the 5% lowest GFP (GFP-low) subpopulations. (A) SOX2 levels of GFP-high and GFP-low subpopulations were quantitated by flow cytometry. (B) The GFP-high and GFP-low subpopulations were seeded in low-attachment plates for mammosphere assays and analysed after 5 days. (C) The GFP-high and GFP-low subpopulations were plated in complete DMEM medium and incubated with the Cell-Titer-Glo 2.0 Reagent for 15 min to determine the ATP levels. Also, two EMT markers were analysed. More specifically, (D) E-cadherin was analysed by Western blotting. β-actin was used as a protein loading control. Independently, (E) Vimentin levels were determined with the Biotracker TiY Vimentin Dye, by flow cytometry. 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.