Fig. 3

EIF5A2 positively modulates stem-like properties in ovarian cancer cells. SKOV3 and HO-8910 cells were transfected with shEIF5A2 or EIF5A2 plasmid, and cultured in serum-free culture condition for 7 days. a Morphology of spheroids derived from SKOV3, Hey, and HO-8910 cells are shown (bar = 40um). b The expression of EIF5A2 and stem-related markers, OCT-4 and ALDH1A1, were detected by Western blotting analysis. c The proportion of CD44+CD24− and CD133+ phenotype in SKOV3 spheroids and HO-8910 spheroids was analyzed by FCM (Figure S5 Quantitative analysis of Flow Cytometry). d Single-cell suspensions with 3000 cells were seeded in 6-well culture plates and cultured in semi-solid serum-free medium for 5 days. The number and size of spheroids formed was determined via microscopy, and representative pictures are shown. Representative images and the numbers of NC, EIF5A2-KD SKOV3 and vector, EIF5A2-OE HO-8910 cell-derived spheroids from three serial passages were compared (Figure S6 Quantitative analysis of spheroid formation ability). e NC, EIF5A2-KD SKOV3 and vector, EIF5A2-OE HO-8910 cells were treated with cis-platinum for 36 h. The cell viability was measured through a CCK-8 assay, and the data are presented as the fold change relative to the treatment-free groups (left: cell viability curve, right: IC50 quantitative analysis). The effect of EIF5A2 knockdown on the drug sensitivity of ovarian cancer cells in vivo was analyzed by inoculating SKOV3 cells into nude mice. Data represents the mean ± standard error of three independent experiments. f Immunofluorescent staining of CD133 (red), EIF5A2 (green) and their co-localization (yellow) in NC and EIF5A2-KD SKOV3-spheroids. g SKOV3 cells were transfected with shNC or shEIF5A2 for 48 h, cultured in spheroid culture conditions for 7 days, and then injected into BALB/c mice (female, 4-week-old) (n = 5 in each group). All mice were sacrificed at week 5 and the tumor incidence was evaluated. Subcutaneous tumors are shown. h Western blot assay were used to detect the protein levels of EIF5A2 and CD133 in the formed tumors. All the data represent the means ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001