Figure 3.
Loss of CYRI controls lamellipodia spread of macrophages. (A) Schematic overview of the cyri gene locus. Exons encoding parts of the DUF1394 domain are highlighted in yellow. The target sequence for CRISPR/Cas9 gene modification and generated cyri deletions, cyriΔ2 and cyriΔ11 is depicted. (B) Loss of cyri mutants were validated by western blot analysis using a specific anti-CYRI antibody. Lysates from ten ovaries of wild type and different mutant flies were analyzed. Anti-tubulin signal served as a loading control. (C–E) Confocal images of (C) wild type; (D) cyri RNAi depleted (E) CYRI-WT overexpressing macrophages stained for endogenous WAVE (α-WAVE; green), F-actin (grey), and DAPI (blue). Scale bars represent 10 µm. (F) Quantification of spread cell area of pre-pupal hemocytes (wild type: 30 cells; cyri RNAi #1: 89 cells; cyri RNAi #2: 85 cells.). Statistical significance was evaluated using one-way-ANOVA (Kruskal–Wallis test) followed by Dunn’s Multiple Comparison test. P value: <0.0001 (***). The red bar represents the median. Three independent transfection experiments were performed. (G) Quantification of immunofluorescent anti-WAVE intensity at the leading edge of wild type (n = 30 cells), cyri RNAi depleted macrophages (two different RNAi transgenes #1 and #2; each n = 29 cells) and macrophages overexpressing a wild type CYRI transgenes (CYRI-WT OE, n = 30 cells) normalized to background fluorescence. One-way-ANOVA test was performed. For multiple comparison, the test was corrected after Dunnett. P = *(0.033), **(0.002), ***(0.001). Quantification was done from three independent experiments. Source data are available for this figure: SourceData F3.

Loss of CYRI controls lamellipodia spread of macrophages. (A) Schematic overview of the cyri gene locus. Exons encoding parts of the DUF1394 domain are highlighted in yellow. The target sequence for CRISPR/Cas9 gene modification and generated cyri deletions, cyriΔ2 and cyriΔ11 is depicted. (B) Loss of cyri mutants were validated by western blot analysis using a specific anti-CYRI antibody. Lysates from ten ovaries of wild type and different mutant flies were analyzed. Anti-tubulin signal served as a loading control. (C–E) Confocal images of (C) wild type; (D) cyri RNAi depleted (E) CYRI-WT overexpressing macrophages stained for endogenous WAVE (α-WAVE; green), F-actin (grey), and DAPI (blue). Scale bars represent 10 µm. (F) Quantification of spread cell area of pre-pupal hemocytes (wild type: 30 cells; cyri RNAi #1: 89 cells; cyri RNAi #2: 85 cells.). Statistical significance was evaluated using one-way-ANOVA (Kruskal–Wallis test) followed by Dunn’s Multiple Comparison test. P value: <0.0001 (***). The red bar represents the median. Three independent transfection experiments were performed. (G) Quantification of immunofluorescent anti-WAVE intensity at the leading edge of wild type (n = 30 cells), cyri RNAi depleted macrophages (two different RNAi transgenes #1 and #2; each n = 29 cells) and macrophages overexpressing a wild type CYRI transgenes (CYRI-WT OE, n = 30 cells) normalized to background fluorescence. One-way-ANOVA test was performed. For multiple comparison, the test was corrected after Dunnett. P = *(0.033), **(0.002), ***(0.001). Quantification was done from three independent experiments. Source data are available for this figure: SourceData F3.

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