Figure 2.

GnT-III expression maintains the SP and promotes spheroid formation in ovarian cancer cells. A, stable OVCAR3 control shRNA and OVCAR3 GnT-III shRNA cells were labeled with Hoechst 33342 dye and analyzed by flow cytometry before and after verapamil treatment. Representative results shown from n = 3 experiments. B, OVCAR3 and OVCA26 cell lines generated 3D independent, self-renewing spheroid cells under stem cell selection medium. Upper row, lower magnification; lower row, zoom in on individual spheroid. Bar, 100 μm. C, cumulative spheroids formed were counted, and data from n = 3 experiments are shown as a percentage. *, p < 0.05. D, spheroid growth and formation were evaluated by dispersing cells from spheroids that had been grown in ultralow adhesion plates in stem cell selective media for 3 weeks. Spheroid density was calculated using ImageJ from five fields for each sample at 2, 24, 48, and 72 h after seeding. The data shown are results from three independent experiments with the density of the OVCA26 GnT-III ShRNA spheroids being set at 1.0. *, p < 0.05; **, p < 0.005. E, total RNA isolated from OVCAR3 spheroid cells were examined for expression of various stemness genes as indicated (primer sequences in supplemental Table S2). Relative expression levels normalized to RPL4 are shown for GnT-III shRNA levels compared with control shRNA set to 1.0 for comparison. The data are means ± S.E., n = 3. *, p < 0.05; **, p < 0.01. F, total RNA isolated from OVCA26 spheroid cells were examined for expression of various stemness genes as indicated (primer sequences in supplemental Table S2). Relative expression levels normalized to RPL4 are shown for GnT-III shRNA levels compared with control shRNA set to 1.0 for comparison. The data are means ± S.E., n = 3. *, p < 0.05.