Figure 4.
Figure 4. Cell-in-cell invasion requires MRTF–SRF and Ezrin. (A) MCF10A cells stably expressing H2B-mCherry together with Ezrin-GFP were imaged during the process of entotic invasion. Note redistribution and enrichment of Ezrin-GFP at the rear of the invading cell during advanced stages of invasion. (B) MCF10A cells stably expressing either Ezrin-mCherry or Ezrin-GFP were mixed and imaged during the process of entotic invasion. Note that the redistribution and enrichment of Ezrin specifically originates from the invading cell. (C) Immunolabeling of endogenous Ezrin (red), phospho-Ezrin/Radixin/Moesin (green), and nuclei (DAPI) of MCF10A cells. Before fixation, cells were cultured for 4 h in suspension to promote entotic invasion. Examples illustrate the enrichment of Ezrin as well as p-ERM at the rear of the invading cell at different stages of the process. Bars, 5 µm. (D) Quantification of entotic invasion after 4 h of culture in suspension. To test for the effects of acute transcriptional inhibition, 50 µg/ml actinomycin D was added to the cells immediately after plating them on Ultra-Low attachment dishes. Entotic events were counted from three different experiments considering ≥2,000 cells for each condition. (E) Western blot confirming efficient siRNA-mediated knockdown of SRF, Ezrin, or MRTF-A together with MRTF-B. Tubulin served as a loading control. (F) Quantification of entotic invasion of cells treated with the indicated siRNAs for 72 h. Entotic events were counted from three independent experiments considering ≥800 cells for each condition. (G) Cells either expressing H2B-mCherry or H2B-GFP were treated with either control or siRNA directed against SRF as indicated, mixed, and allowed to undergo entotic invasion. Resulting entotic events were considered (100%) and scored according to the underlying color combinations as indicated. Note the significantly impaired invasion of SRF-silenced cells. ≥290 entotic events were scored from three independent experiments. Error bars indicate SD. Asterisks indicate statistical significance (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). ns indicates no significance (P > 0.05).

Cell-in-cell invasion requires MRTF–SRF and Ezrin. (A) MCF10A cells stably expressing H2B-mCherry together with Ezrin-GFP were imaged during the process of entotic invasion. Note redistribution and enrichment of Ezrin-GFP at the rear of the invading cell during advanced stages of invasion. (B) MCF10A cells stably expressing either Ezrin-mCherry or Ezrin-GFP were mixed and imaged during the process of entotic invasion. Note that the redistribution and enrichment of Ezrin specifically originates from the invading cell. (C) Immunolabeling of endogenous Ezrin (red), phospho-Ezrin/Radixin/Moesin (green), and nuclei (DAPI) of MCF10A cells. Before fixation, cells were cultured for 4 h in suspension to promote entotic invasion. Examples illustrate the enrichment of Ezrin as well as p-ERM at the rear of the invading cell at different stages of the process. Bars, 5 µm. (D) Quantification of entotic invasion after 4 h of culture in suspension. To test for the effects of acute transcriptional inhibition, 50 µg/ml actinomycin D was added to the cells immediately after plating them on Ultra-Low attachment dishes. Entotic events were counted from three different experiments considering ≥2,000 cells for each condition. (E) Western blot confirming efficient siRNA-mediated knockdown of SRF, Ezrin, or MRTF-A together with MRTF-B. Tubulin served as a loading control. (F) Quantification of entotic invasion of cells treated with the indicated siRNAs for 72 h. Entotic events were counted from three independent experiments considering ≥800 cells for each condition. (G) Cells either expressing H2B-mCherry or H2B-GFP were treated with either control or siRNA directed against SRF as indicated, mixed, and allowed to undergo entotic invasion. Resulting entotic events were considered (100%) and scored according to the underlying color combinations as indicated. Note the significantly impaired invasion of SRF-silenced cells. ≥290 entotic events were scored from three independent experiments. Error bars indicate SD. Asterisks indicate statistical significance (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). ns indicates no significance (P > 0.05).

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