Fig. 3

As SP cells, CDC50A-positive SKOV3 cells show similar characteristics to EOC initial cells. A CDC50A⁺ SKOV3 cells were sorted and cultured in vitro, and most of them differentiated into CDC50A⁻ cells after 2 weeks, while CDC50A⁻ SKOV3 cells could not generate CDC50A⁺ cells. This demonstrated the ability of CDC50A + SKOV3 cells to differentiate. B 1000 cells were cultured for sphere forming. After 7 days, isolated CDC50A⁺ SKOV3 cells generated 2–3 times more spheres than CDC50A⁻ cells (**p < 0.001). CDC50A⁻SKOV3 cells exhibited cell adhesion with each other but not spheres. C A higher ratio of CDC50A⁺ SKOV3 cells was detected in spheres than in adherent cultured cells through both immunofluorescence and FACS. Scale bar was 50 μm. D The numbers of spheres decreased significantly after the expression of CDC50A was downregulated through shRNA (**p < 0.001). E Through qRT-PCR, the mRNA expressions of some stem cell markers, such as Bmi-1, β-catenin, APC, E-cadherin, Vimentin, Notch-1 and Oct-4, increased significantly in CDC50A⁺ OVCAR4 (all p < 0.05). F The expressions of above stem cell markers were detected using Western-blot, and all of them increased significantly, including TGF-β1 of which the mRNA level was the same between CDC50A⁺ and CDC50A⁻ OVCAR4. Gray value of bands was measured using ImageJ software (National Institutes of Health). The grouping of blots cropped from different gels. The blots were cut prior to hybridisation with antibodies. The images were generated by chemiluminescence image analysis system (Tanon 520, Shanghai, China). The raw data with detail description was shown in Supplementary Figs. 7 and 8