Fig. 1: Ascites-derived tumour cells and tumourspheres show CSC properties and express higher levels of PDK4.

a Transwell migration/invasion and sphere formation assays of cancer cells derived from primary ovarian tumours and ascites. (Left) Representative images of migrating or invading cells (scale bar, 100 μΜ) and sphere formation (scale bar, 50 μΜ). (Middle) Cell migration and invasion are presented as a percentage of ascites. (Right) Quantification of cells forming tumourspheres. b Flow cytometry analysis of ALDH and CD44 in single cells isolated from primary ovarian tumour or ascites. c Relative PDK4 expression in HOSE 96-9-18 and cancer cells derived from primary ovarian tumours and ascites determined via qPCR. d qPCR analysis of relative stemness genes, NANOG, OCT4, SOX2, KLF4 and BMI1, of tumourspheres formed from ascites-derived tumour cells and ovarian cancer cell lines (SKOV3 and OVCAR3), compared to monolayer cells. e (Upper) Relative mRNA expression of PDK 1–4 in tumourspheres and monolayer cells isolated from SKOV3 cells determined using qPCR. (Lower) Relative PDK4 mRNA expression in tumourspheres and monolayer cells isolated from ascites, SKOV3 and OVCAR3 cells. f Immunoblotting showing protein expression of OCT4, SOX2, KLF4, BMI1 and PDK4 of tumourspheres and monolayer cells isolated from SKOV3 cells. g Immunoblotting showing PDK4 expression in tumourspheres and monolayer cells isolated from ascites cancer cells and OVCAR3 cells. h Relative lactate production of tumourspheres and monolayer cells isolated from ascites, SKOV3 and OVCAR3 cells after 24 h incubation assessed using Lactate Colorimetric Assay Kit II (*p < 0.05, **p < 0.01, results represent means ± SD from three independent experiments; n.s., not significant).