Fig. 5

Induction of EMT in MCF10A cells overexpressing TWIST or SNAI2. The EMT-linked transcription factors TWIST and SNAI2 were overexpressed in epithelial MCF10A cells. EMT was manifested by acquisition of mesenchymal traits. a Fluorescence microscopy for detection of vimentin and E-cadherin, and cell morphology (using phalloidin to stain F-actin), in the parental cells (MCF10A/Par), and cells overexpressing TWIST (MCF10A/TWIST) and SNAI2 (MCF10A/SNAI2). b Images (phase-contrast microscopy) showing spheroid formation capacity. c Total cellular RNA versus DNA content (Hoechst 33258) in MCF10A/TWIST, compared to MCF10A/Par (flow cytometry). d Protein expression of subunit SDHB and SDHC in MCF10A/Par and MCF10A/TWIST cells. e Mitochondrial respiration after overexpression of EMT-linked transcription factors. Oxygen consumption rate (OCR) was measured after sequential additions of oligomycin (Oligo), CCCP, rotenone (Rot), and antimycin A (AMA), in DMEM medium. f Extracted data from the experiment in (e), showing rates of basal and leak (with oligomycin) respiration and respiratory capacity (uncoupled, with CCCP), in the MCF10A/TWIST and MCF10A/SNAI2 cells relative to parental cells (CTR). g Leak respiration (with oligomycin) as the percentage of respiratory capacity (uncoupled, with CCCP), from the experiment in (e). h SDH activity measured in restricted medium (MAS) after the supply of rotenone (Rot), succinate (Succ), ADP, and permabilizing agent (PMP). Oligomycin (Oligo) and antimycin A (AMA) were then added to control mitochondrial integrity and background activity. i The diagram shows statistical data from the experiment described in (h). Data are shown as mean ± SD (column plots) or mean ± SEM (OCR traces). Student’s t test was used for statistical analysis. *p < 0.01