Figure 5.
Depletion of Rab18 affects cell spreading and architecture on adhesive micropatterns. (a) U2OS cells transfected with siRNA control, siRNA Rab18, or siRNA Rab18_1, or treated with the same siRNAs against Rab18 and afterward transfected with GFP-Rab18, were plated on fibronectin-coated coverslips and left to adhere for 1 h before fixation and staining with rhodamine-conjugated phalloidin. Transmission images are shown in the lower panel. Scale bar: 20 µm. (b) Quantification of the average area. The graph represents the mean ± SEM; n > 80 cells per experiment for three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (c) Cell lysates from control cells, cells silenced for Rab18, and cells silenced for Rab18 and transfected with GFP-Rab18 were subjected to Western blot analysis with antibodies against Rab18 and tubulin (as loading control). (d) U2OS cells treated with siRNA control, siRNA Rab18, or siRNA Rab18_1 were plated onto fibronectin-coated L-shaped micropatterns and left to adhere for 3.5 h before fixation and staining with rhodamine-conjugated phalloidin. The cells silenced for Rab18 show two different phenotypes: collapsed along the hypotenuse (top) or unable to fully spread on the micropattern (lower). The L-shaped micropattern is shown in red. Scale bar: 10 µm. (e) The percentage of cells with straight or curved hypotenuse (mean ± SEM). n > 80 cells from three independent experiments. **, P < 0.01; ***, P < 0.001. (f) Color-coded map of the actin distribution. The images were obtained by using the CellRef macro on averaged Z-projection images from aligned single stacks. Scale bar: 10 µm.

Depletion of Rab18 affects cell spreading and architecture on adhesive micropatterns. (a) U2OS cells transfected with siRNA control, siRNA Rab18, or siRNA Rab18_1, or treated with the same siRNAs against Rab18 and afterward transfected with GFP-Rab18, were plated on fibronectin-coated coverslips and left to adhere for 1 h before fixation and staining with rhodamine-conjugated phalloidin. Transmission images are shown in the lower panel. Scale bar: 20 µm. (b) Quantification of the average area. The graph represents the mean ± SEM; n > 80 cells per experiment for three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (c) Cell lysates from control cells, cells silenced for Rab18, and cells silenced for Rab18 and transfected with GFP-Rab18 were subjected to Western blot analysis with antibodies against Rab18 and tubulin (as loading control). (d) U2OS cells treated with siRNA control, siRNA Rab18, or siRNA Rab18_1 were plated onto fibronectin-coated L-shaped micropatterns and left to adhere for 3.5 h before fixation and staining with rhodamine-conjugated phalloidin. The cells silenced for Rab18 show two different phenotypes: collapsed along the hypotenuse (top) or unable to fully spread on the micropattern (lower). The L-shaped micropattern is shown in red. Scale bar: 10 µm. (e) The percentage of cells with straight or curved hypotenuse (mean ± SEM). n > 80 cells from three independent experiments. **, P < 0.01; ***, P < 0.001. (f) Color-coded map of the actin distribution. The images were obtained by using the CellRef macro on averaged Z-projection images from aligned single stacks. Scale bar: 10 µm.

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