Figure 4. The PBR of ARL4C is required for ARL4C and IQGAP1 binding.

(A) A schematic representation of four ARL4C-GFP mutants is shown. (B) S2-CP8 cells were transfected with the indicated mutants of ARL4C-GFP. The percentages of cells with ARL4C-GFP mutant accumulated at invasive pseudopods compared with the total number of cells were calculated. (C,D) Lysates of X293T cells expressing the indicated proteins were immunoprecipitated with anti-GFP antibody and the immunoprecipitates were probed with anti-HA and anti-GFP antibodies. (E) S2-CP8 WT or ARL4C KO cells transfected with control or the indicated mutants of ARL4C-GFP were stained with anti-IQGAP1 antibody and phalloidin. The percentages of cells with IQGAP1 accumulated at invasive pseudopods compared with the total number of cells were calculated. (F) S2-CP8 WT or IQGAP1 KO cells were transfected with ARL4C-GFP. The percentages of cells with ARL4C-GFP accumulated at invasive pseudopods compared with the total number of cells were calculated. (G) S2-CP8 cells stably expressing GFP or the indicated mutants of ARL4C-GFP were transfected with control or ARL4C ASO and subjected to invasion assays. Invasive ability is expressed as the percentage of the same cells transfected with control ASO. (B,E–G) Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using a two-tailed Student’s t-test (F,G) or one-way ANOVA followed by Bonferroni post hoc test (B,E). (B,E,F) The regions in the yellow dashed squares are shown enlarged in the left bottom images. The right bottom images are shown in a false color representation of fluorescence intensity. False color representations were color-coded on the spectrum. Scale bars in (B,E,F) 10 μm. KO, knockout. RFI, relative fluorescence intensity. n.s., not significant. *, p < 0.05; **, p < 0.01. See Figure 4—source data 1.

Figure 4—figure supplement 1. ARL4C is essential for recruitment of IQGAP1 to invasive pseudopods.

(A-C) S2-CP8 cells were transfected with the indicated mutants of ARL4C-GFP and stained with the indicated antibodies. The percentages of cells with invasive pseudopods compared with the total number of cells were calculated (B). Cells were classified according to the number of pseudopods as indicated (C). (D) S2-CP8 WT or ARL4C KO cells were stained with anti-IQGAP1 antibody and phalloidin. Circles of 2 μm diameter were placed at the edge of pseudopods and mean intensity of each circle was measured. The distribution of the data was depicted as a violin plot and the center lines show the medians. More than 100 pseudopods were calculated for each condition. p Values were calculated using a two-tailed Student’s t-test. (E-G) S2-CP8 WT or IQGAP1 KO cells were stained with the indicated antibodies and the same assays as in (B) and (C) were performed for (F) and (G), respectively. (H) S2-CP8 WT cells transfected with control ASO or ARL4C ASO-1316 were stained with anti-IQGAP1 antibody and phalloidin. The percentages of cells with IQGAP1 accumulated at invasive pseudopods compared with the total number of cells were calculated. (I) S2-CP8 cells expressing GFP or the indicated mutants of ARL4C-GFP were transfected with control or ARL4C ASO-1316. Lysates were probed with the indicated antibodies. (A,D,E,H) The regions in the yellow dashed squares are shown enlarged in the left bottom images. The right bottom images are shown in a false color representation of fluorescence intensity. False color representations were color-coded on the spectrum. (B,F,H) Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using a two-tailed Student’s t-test (F,H) or one-way ANOVA followed by Bonferroni post hoc test (B). Scale bars in (A,D,E,H) 10 μm. KO, knockout. RFI, relative fluorescence intensity. n.s., not significant. **, p < 0.01. See Figure 4—figure supplement 1—source data 1.