Fig. 3
Induction of EMT in MCF7 upon SDHC knockdown. Parental MCF7 cells (MCF7 SDHC+/+) were modified by CRISPR/Cas9 editing to knock down the expression of SDHC (MCF7 SDHC+/−). a SDHA-D mRNA was analyzed by qPCR. b SDHC protein expression was analyzed by western blotting. c Confocal microscopy was performed to evaluate E-cad (immuno-stained, green) expression level and cell morphology (F-actin stained by phalloidin, red). d mRNA expression of the EMT markers E-cad (CDH1), vimentin (VIM), TWIST1, SNAI2, and Axl. e Spheroid formation (anchorage-independent) was evaluated after seeding the cells in wells with low surface adherence. f Spheroid growth and stability was assessed after centrifugation-aided spheroid formation. The spheroid size was measured after 48 h in culture. g The diagram shows statistical data from the experiment described in (f). h Mitochondrial respiratory rates were measured in MCF7 SDHC+/+ and MCF7 SDHC+/− cultures, with glucose, pyruvate, and glutamine provided as the major fuels. Oxygen consumption rate (OCR) was monitored upon sequential additions of oligomycin (O, 3 μM), CCCP (C, 0.75 μM), rotenone (R, 1 μM), and antimycin A (A, 1 μM) as indicated, to assess specific properties of mitochondrial respiration. i For measurement of SDH-dependent mitochondrial respiration, the cells were permeabilized (with PMP) and rotenone was added prior to analysis in restricted assay medium (MAS). Succinate (SUCC, 10 mM), ADP (4 mM), oligomycin (OLIGO, 3 μM), and antimycin A (AMA, 1 μM) were added sequentially as indicated. j Fluorescence microscopy was performed to compare cell morphology (F-actin stained by phalloidin, white) in MCF7 SDHD/C+/+, MCF7 SDHC+/−, and MCF7 SDHD+/− cultures. k Scratch-wound assay comparing MCF7 SDHD/C+/+, MCF7 SDHC+/−, and MCF7 SDHD+/− cells. The images were taken 24 h after the scratch was made. l In the experiment described in (k), we measured scratch size as gap distance (d) at a fixed position, after 24 h, and calculated the results relative to the initial scratch size. Each dot represents separate wells. Data are shown as mean ± SD for (a), (d), (g), and (l) and mean ± SEM for (h) and (i). Student’s t test was used for statistical analysis. *p < 0.01; ns, not significant