Fig. 4
Metastatic ovarian cancer cells enhance c-Kit, Notch3, and membrane raft-associated PHB and contribute to CSC enrichment. a Production of metastasized ovarian cancer cells from several passages of SKOV3GL cells into the mouse intraperitoneal cavity. The migration ability increased in SKOV3GL-G3 and SKOV3GL-G4 cells as determined by a 24-h Boyden chamber assay. b The levels of phospho-PHBY259, phospho-PHBT258, and PHB in the lipid raft or cytosolic plus non-raft membrane (C + M) fraction of SKOV3GL, SKOV3GL-G1 to -G4 cells were detected by western blotting. c Expression of c-Kit, full-length (FL) Notch3 and membrane tethered Notch3 fragment (NTM) were analyzed from SKOV3GL to SKOV3GL-G4 cells by western blotting. d SKOV3GL-G4 cells were transfected with scrambled control or c-Kit siRNA for 48 h. Total cellular c-Kit, Notch3 (FL & NTM), phospho-PHB, PHB and β-actin protein expression was analyzed by western blotting. e Colony forming assay. SKOV3GL and SKOV3GL-G1 to -G4 cells were plated at a density of 300 cells/well in 6-well plates. f Phase contrast photomicrographs showing clonal expansion of SKOV3GL-G4 cells into the oncosphere over a 12-day period (top). Photomicrographs of SKOV3GL-G4 adherent cells, oncosphere expansion in low-adherence culture, and redifferentiated oncosphere cells after return to adherent culture (middle). qPCR for putative stem cell markers (Oct4, Nanog, SOX2) expressed as fold of SKOV3GL parental cells (bottom). Scale bar = 50 μm. All statistics represent the mean ± SD of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05, t test. Blots are representative of three independent experiments. g SKOV3GL and SKOV3GL-G4 cells were seeded in CSC medium for 12 days. After 12 days, spheres were harvested and trypsinized into single cells. A total of 1000 or 100,000 cells in 50 μl DPBS were mixed with 50 μl Matrigel and subcutaneously injected into the flanks of nude mice. In vivo CSC properties were determined by luciferase signal at week 4 post injection (mean ± SD; n = 3 per group; **, P < 0.01; *, P < 0.05, t test)