Figure 1.

Effect of CA9 silencing on the expression of ROCK1 protein and initial cell adhesion and spreading. (A) HT1080 cells were pre-incubated with or without doxycycline (±DOX) to induce expression of shRNA. Cells were then trypsinized and placed to hypoxia (2% O₂) for 24 h. The level of ROCK1 protein was reduced in the CA9-silenced cells. Control HT1080 Scr shRNA had the same level of ROCK1 as non-silenced CA9 shRNA (–DOX). The addition of doxycycline to HT1080 Scr shRNA cells did not cause any change in CA IX protein level, nor in ROCK1 expression. HeLa cells transiently silenced for CA9 (pSUPER-shCA9) also showed a decreased level of ROCK1 protein compared to control cells (ctrl, pSUPER-shScr). (B,C) Time-lapse analysis of the entire population (B) and individual cells (C) of CA9-silenced and control HT1080 cells. (B) CA9-silenced cells (–CA IX) exhibited significantly smaller spreading areas than control non-silenced cells (+CA IX). Graph shows the mean ± standard deviation values for at least 200 cells, significance of differences among samples was tested by t-test (^**p < 0.01). (C) In the analysis of individual cells, HT1080 expressing CA IX show faster spreading and achieve larger spreading area when compared with silenced cells (–CA IX). The graph of circularity demonstrates that silenced cells preserve a circular morphology for a longer amount of time, CA IX positive cells, on the opposite, became more polarized. Graphs give mean ± standard deviation values for designated time-points for 29 CA IX expressing cells and 39 silenced cells (–CA IX). (D) Representative pictures of characteristic morphology of CA9-silenced (–CA IX) and control cells (–CA IX) during the process of initial adhesion and spreading at given time-points. Scale bar 20 μm.