Figure 5.

Ezrin expression restores membrane blebbing and entotic invasion in SRF-silenced cells. (A) Western blot confirming efficient siRNA-mediated knockdown of SRF in cells stably expressing either GFP or Ezrin-GFP. Tubulin served as a loading control. (B) Live MCF10A cells stably expressing either GFP or Ezrin-GFP were imaged over time to visualize PM blebbing. Bars, 5 µm. (C) Quantification of maximum bleb expansion in cells stably expressing Ezrin-GFP. Cells were treated with the indicated siRNAs, and the maximum length reached by individual blebs was compared. Note no significant differences between control and SRF-silenced cells in contrast to cells only expressing GFP (Fig. 1 D). (D) Cells stably expressing Ezrin-GFP were monitored to measure the time for expansion and retraction of individual blebs. Before imaging, cells were treated with siRNA as indicated. Note no significant differences between control and SRF-silenced cells in contrast to cells expressing GFP only (Fig. 1 E). (C and D) ≥60 blebs and 15 cells per condition. ns indicates no significance (P > 0.05). (E) Quantification of entotic invasion of cells stably expressing GFP, Ezrin-GFP, or Ezrin-T567A-GFP. Cells were transfected with either control or siRNA against SRF as indicated, and the number of entotic events was quantified from three independent experiments considering ≥800 cells as a percentage of the total number of cells. Error bars indicate SD. Asterisks indicate statistical significance (****, P ≤ 0.0001). ns indicates no significance (P > 0.05).