Figure 3.

Resveratrol induces dormant-like cell cycle arrest in ovarian cancer spheroids exposed to IL-6. A-B-C. OVCAR3 cells were grown as 3D spheroids and treated following the same timeline as Figure 2. (A) The assessment of cell proliferation rate of OVCAR3 3D spheroids. On day 10, DiD-stained 3D spheroids were cytospotted on glass slides and immediately observed under a fluorescence microscope. Scale bar = 20 µm; magnification = 63×. (B) Quantification of DiD dye retention performed by ImageJ software. Data ± S.D. represent three independent replicates. Statistical analysis was performed by using GraphPad Prism 5.0 software. Bonferroni’s multiple comparison test after one-way ANOVA was employed. Significance was considered as follows: **** p < 0.0001; ** p < 0.01. (C) Western blotting for the expression of the proliferation/quiescence markers (ERK1/2, p38, and p21) in 3D OVCAR3 spheroids collected on day 10. The densitometric analysis of Western blotting representative of three independent experiments is included. (D–G). OVCAR3 cells were cultured in 2D and treated as above. (D) Colony formation assay calculated on day 5 and day 10. Data ± S.D. are representative of three independent replicates. (E) Doubling time calculated on day 5 and day 10. Data ± S.D. are representative of three independent replicates. (F) Cell cycle analysis performed on day 5 and day 10 by using a FacScan flow cytometer (G) Immunofluorescence double-staining performed on day 5 and day 10 for ARH-I (red)–Ki-67 (green) and for p21 (red)–cyclin D1 (green). Scale bar = 20 µm; magnification = 63×.