Skip to main content
. 2021 Sep 30;10:e66721. doi: 10.7554/eLife.66721

Figure 2. ARL4C expression is especially involved in the invasion of pancreatic cancer cells.

(A-D) S2-CP8 cells (A,B) or S2-CP8 cells expressing GFP or ARL4C-GFP (C,D) were transfected with control or ARL4C ASO-1316 and subjected to migration (A,C) and invasion (B,D) assays. Migratory and invasive abilities are expressed as the percentage of the same cells transfected with control ASO. (E) A schematic illustration of 3D invasion into collagen I gel using a 3D cell culture chip is shown. There is a chemical concentration gradient across the gel channel and cells can invade into the gel. The right panel shows a fluorescent confocal image (top) and a 3D reconstructed image (bottom). (F) S2-CP8 cells were transfected with control or ARL4C ASO-1316 and subjected to a 3D collagen I gel (2 mg/mL) invasion assay. The distances from the edge of the gel interface of all cells invading into the collagen gel were measured. (G) The same assay as in (F) was performed in the presence of different concentrations of collagen I. (H) S2-CP8 cells stably expressing ARL4C-tdTomato were observed with time-lapse imaging. Arrowheads indicate the tips of invasive pseudopods and yellow circles indicate the cytoplasm (20 μm away from the tip of pseudopods). The region in the yellow dashed squares is shown enlarged in the bottom image. Fluorescence intensities of the cytoplasm and invasive pseudopods were measured and plotted as a function of time. (I) S2-CP8 cells were subjected to a 3D collagen I gel invasion assay and stained with phalloidin and Hoechst 33342. The angle of pseudopods to the direction of cell invasion toward FBS was calculated (n = 105). The results were plotted to a polar histogram. (J) S2-CP8 cells expressing ARL4C-tdTomato were subjected to a 3D collagen I gel invasion assay with DQcollagen I, and stained with phalloidin and Hoechst 33342. The regions in the yellow dashed squares (a, pseudopod; b, cell body) are enlarged. (K) S2-CP8 cells transfected with control ASO or ARL4C ASO-1316 were subjected to a 3D collagen I gel invasion assay with DQcollagen I. The percentages of cells with DQcollagen I-positive pseudopods compared with the total number of cells were calculated. (A–D,F,G,K) Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using a two-tailed Student’s t-test. Scale bars in (F,G) 100 μm; (H) 20 µm; (J) 10 µm; (K) 5 µm. n.s. not significant. *, p < 0.05; **, p < 0.01. See Figure 2—source data 1.

Figure 2—source data 1. Excel file containing quantitative data for Figure 2.

Figure 2.

Figure 2—figure supplement 1. ARL4C expression is involved in invasion of pancreatic cancer cells rather than in sphere formation.

Figure 2—figure supplement 1.

(A) S2-CP8 cells transfected with control or ARL4C ASO-1316 were cultured for 6 days in 2.5D Matrigel. The cells were then fixed and stained with phalloidin and Hoechst 33342 and sphere areas were calculated. When more than 10 cells formed a spherical structure, it was counted as one sphere. Data are shown as a box and whiskers plot. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers indicate the smallest and largest values; dots show all of the individual values. More than 65 spheres were analyzed per group. p Values were calculated using a two-tailed Student’s t-test. (B) Lysates were prepared from S2-CP8 cells transfected with control or ARL4C ASO-1316 and probed with the indicated antibodies. (C) S2-CP8 cells treated with control ASO, ARL4C ASO-1316, or staurosporine were cultured for 3.5 days and subjected to cytotoxic assay. Staurosporine was treated 15 hr before the assay. Cells were incubated with propidium iodide (PI) and Hoechst 33342. PI-positive cells are expressed as the percentage of positively stained cells compared with total Hoechst 33342 stained cells per field. (D) PANC-1 cells transfected with control or ARL4C ASOs were subjected to migration and invasion assays. Migratory and invasive abilities are expressed as the percentage of control cells. (E) S2-CP8 cells stably expressing GFP or ARL4C-GFP were transfected with control or ARL4C ASO-1316. Lysates were probed with the indicated antibodies. (F) S2-CP8 cells stably expressing ARL4C-tdTomato were stained with the indicated antibodies. The regions in the yellow dashed squares are shown enlarged in the bottom images. (G) S2-CP8 cells were transfected with the indicated ARL4C-GFP mutants and stained with phalloidin. 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. The percentages of cells with ARL4C-GFP mutant accumulated at invasive pseudopods compared with the total number of cells were calculated. False color representations were color-coded on the spectrum. (C,D,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 (D) or one-way ANOVA followed by Bonferroni post hoc test (C,G). Scale bars in (A) 50 μm; (C) 100 μm; (F,G) 10 μm. RFI, relative fluorescence intensity. n.s., not significant. *, p < 0.05; **, p < 0.01. See Figure 2—figure supplement 1—source data 1.
Figure 2—figure supplement 1—source data 1. Excel file containing quantitative data for Figure 2—figure supplement 1.
Figure 2—figure supplement 2. ARL4C localizes at the tips of invasive pseudopods.

Figure 2—figure supplement 2.

(A) S2-CP8, PANC-1, and BxPC-3 cells were subjected to an invadopodia assay and then stained with phalloidin. (B) S2-CP8 cells were stained with the indicated antibodies. (C,D) S2-CP8 cells treated with control ASO or ARL4C ASO-1316, and S2-CP8 WT or ARL4C KO cells were stained with anti-cortactin antibody and phalloidin. The percentages of cells with invasive pseudopods compared with the total number of cells were calculated (C). Cells were classified according to the number of pseudopods as indicated (D). (E) S2-CP8 cells were transfected with control or ARPC2 siRNAs, and ARPC2 mRNA levels were measured by quantitative real-time PCR. Relative ARPC2 mRNA levels were normalized to those of GAPDH and expressed as fold changes compared with the level in control cell. (F,G) The same assays as in (C) and (D) were performed for S2-CP8 cells transfected with control or ARPC2 siRNAs. (H,I) S2-CP8 WT or ARL4C KO cells were stained with cortactin or ARPC2. 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. (B,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. (C,E,F) Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using a two-tailed Student’s t-test (C) or one-way ANOVA followed by Bonferroni post hoc test (E,F). Scale bars in (A,B,H) 10 μm. au, arbitrary units. KO, knockout. RFI, relative fluorescence intensity. n.s., not significant. *, p < 0.05; **, p < 0.01. See Figure 2—figure supplement 2—source data 1.
Figure 2—figure supplement 2—source data 1. Excel file containing quantitative data for Figure 2—figure supplement 2.
Figure 2—figure supplement 3. ARL4C is involved in invasion into 3D matrix.

Figure 2—figure supplement 3.

(A) S2-CP8 cells were subjected to a 3D collagen I gel invasion assay and the image shows cells, indicated by black dashed square, placed at the starting position of this assay at 0 time. (B) S2-CP8 WT or ARL4C KO cells were subjected to a 3D collagen I gel (2 mg/mL) invasion assay. The distances from the edge of the gel interface of all cells invading into the collagen gel were measured. Data are shown as the mean ± s.d. of three biological replicates. p Values were calculated using a two-tailed Student’s t-test. Scale bar in (B) 100 μm. KO, knockout. **, p < 0.01. See Figure 2—figure supplement 3—source data 1.
Figure 2—figure supplement 3—source data 1. Excel file containing quantitative data for Figure 2—figure supplement 2.
Figure 2—video 1. ARL4C accumulates at the tips of invasive pseudopods.
Download video file (253KB, mp4)
S2-CP8 cells stably expressing ARL4C-tdTomato were subjected to the 3D collagen I gel invasion assay and were observed with time-lapse imaging and the video was acquired for 78 min. Cells were imaged every 3 min.
Figure 2—video 2. S2-CP8 cells extend pseudopods to the direction of cell invasion.
Download video file (260.3KB, mp4)
S2-CP8 WT cells were subjected to the 3D collagen I gel invasion assay and were observed with time-lapse imaging and the video was acquired for 4 h 35 min. Cells were imaged every 5 min.
HHS Vulnerability Disclosure