Figure 3. Effect of Hic-5 overexpression on A2780s cell proliferation migration, invasion and cell cycle.

(A) Cell proliferation of Hic-5 pCMV clone (pCMV-Hic-5) was compared to the control clone (pCMV-Ctrl). (B) Cell migration of Hic-5 pCMV clone (pCMV-Hic-5) was compared to the control clone (pCMV-Ctrl). Migration was assessed using Boyden-chamber assay. Cells from the Hic-5 pCMV clone (pCMV-Hic-5) and the control clone (pCMV-Ctrl) were seeded into the upper chambers in 0.1% FBS containing medium at a density of 1.5 × 10⁴ per well, and 600 μl of 1% FBS containing medium was placed in the lower chamber as a chemoattractant. After 24 hr incubation at 37°C in 5% CO₂, the cells were fixed with cold methanol and stained with blue trypan solution. Migrated cells on the underside of the filter were photographed and counted by phase contrast microscopy. (C) Cell invasion was assayed in a similar way, as the upper chambers were coated with Matrigel. Here, NIH3T3 conditioned medium was added in the lower chamber as a chemoattractant (see Methods for details). All experiments were performed in triplicates. For each experiment, cell numbers were calculated as the total count from 10 random fields per filter (at magnification × 40). The bar graphs in panels (D) and (E) represent quantitative determinations of migration and invasion data obtained by selecting 10 random fields per filter under phase contrast microscopy and results are expressed as number of cell change (migration and invasion) of the Hic-5 pCMV (pCMV-Hic-5) clone compared to the control clone (pCMV-Ctrl). Differences between pCMV-Hic-5 and pCMV-Ctrl A2780s cells were determined by a Student's t-test; error bars denote mean ± SEM; (p < 0.05). (F) Cell-cycle profile was examined by flow cytometry and percentages of cells in G0/G1, S, and G2/M phase in the Hic-5 pCMV clone (pCMV-Hic-5) were compared to the control clone (pCMV Ctrl). Propidium iodide staining shows a decreased fraction of cells in the G1-phase and an increase accumulation of cells in the S-phase at 0 hr, and 6 hr post hydroxyurea removal in the pCMV-Hic-5 clone, when compared with the pCMV-Ctrl clone.