Figure 2

Knockdown of HOXA9 inhibits aggregation of floating EOC cells, increases anoikis, decreases EOC-mesothelial cell interactions, and reduces EOC cell migration in vitro. (A-D) Cells of control and HOXA9-knockdown SKOV3ip lines were incubated as suspension cultures in polyHEMA-coated plates for 3 days and evaluated for changes in cell morphology and cell death. (A) Cell morphology viewed by phase-contrast light microscopy. Bar, 100 μm. The extent of cell death was evaluated by flow cytometric analysis of (B) 7AAD staining and (C) staining of active caspase-3. In (D), cell death was evaluated by assaying mono- and oligo- nucleosomes in cell lysates by ELISA. Shown are mean ± sd values of three independent experiments. Significance of differences was evaluated by Student t- test. (E,F) Equivalent numbers of control and HOXA9-knockdown SKOV3ip cells that stably expressed GFP were seeded onto confluent monolayers of normal human omental mesothelial cells. At 1 h thereafter, attached tumor cells were viewed by fluorescence microscopy and counted in three random 100× microscopic fields per assay. (E) Representative examples of SKOV3ip cells (shown in green) attached to mesothelial monolayers. Bar, 100 μm. (F) Mean ± sd values of three independent attachment assays. (G) Equivalent numbers of control and HOXA9-knockdown SKOV3ip cells were seeded in transwell chambers. At 6 h thereafter, migrating cells were counted in three random 100x microscopic fields per assay. Shown are mean ± sd values of three independent migration assays.