Figure 4.
ARF3 is a rheostat for the modality of collective invasion. (A–D) PC3 cells expressing mNG, (A) ARF1-mNG, or (C) ARF3-mNG and Scr shRNA in 3D invasion assay. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/2. Scale bars, 100 μm. Magnified images of boxed regions shown. White and black arrowheads, invasive chain or sheet, respectively. RWD at t = Max1/2, normalized to mNG is shown in graphs (B and D). Data are mean ± SEM (3 experimental replicates, triangles, 2–5 technical replicates, circles). P values (Student’s two-tailed t test), **P ≤ 0.01. (E and F) PC3 acini expressing mNG, (E) ARF1-mNG, or (F) ARF3-mNG and Scr shRNA were classified into Round, Spindle, and Spread. n = 6 and 4 experimental replicates for ARF1-mNG and ARF3-mNG, respectively, each with 2–4 technical replicates/condition. (E) 5,005 (mNG), 1,938 (ARF1-mNG) and (F) 9,320 (mNG), 8,699 (ARF3-mNG) mNG-positive acini quantified in total. Heatmaps, Area is mean of Z-score normalized values (purple to yellow). P values, Student’s t test, Bonferroni adjustment, represented by size of bubble. Heatmaps, Round, Spindle, or Spread is Log2 fold change from control (mNG; blue to red). Proportion of control at each time is Z-score normalized (white to black). P values, CMH test, Bonferroni adjusted, represented by size of bubble. Dot indicates P value (Breslow–Day test, Bonferroni-adjusted) for consistent effect magnitude. (G) Schema, Class 1 ARFs share 100% identical core region but differ in seven amino acids (AA) in N and C termini. ARF chimeras with ARF3 N-terminal and ARF1 C-terminal (3N/1C) and ARF3 C-terminal and ARF1 N-terminal (1N/3C) created. (H) Western blot of PC3 cells expressing mNG, ARF1-mNG, ARF3-mNG, and ARF-mNG chimeras for mNG and GAPDH, as loading control. Panels shown are representative of 3 independent lysate preparations. (I) ARF-GTP pulldown and representative Western blot for mNG, GST, and GAPDH, as loading control for both. n = 3 independent lysate preparations and pulldowns. Graphs show mean GGA3 binding ± SEM normalized to ARF3. P values (Student’s two-tailed t test), *P ≤ 0.05. (J and K) PC3 cells expressing mNG, ARF3-mNG, or ARF-mNG chimeras plated in 3D invasion assay. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/2. Scale bars, 100 μm. Magnified images of boxed regions shown. White and black arrowheads, invasive chain or sheet, respectively. RWD at t = Max1/2, normalized to mNG shown in K. Data is mean ± SEM (3 experimental replicates, triangles, 2–5 technical replicates, circles). P values (Student’s two-tailed t test), ***P ≤ 0.001 and ****P ≤ 0.0001. (L) Schema, ARF3 expression levels affect mode of invasion. Source data are available for this figure: SourceData F4.

ARF3 is a rheostat for the modality of collective invasion. (A–D) PC3 cells expressing mNG, (A) ARF1-mNG, or (C) ARF3-mNG and Scr shRNA in 3D invasion assay. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/2. Scale bars, 100 μm. Magnified images of boxed regions shown. White and black arrowheads, invasive chain or sheet, respectively. RWD at t = Max1/2, normalized to mNG is shown in graphs (B and D). Data are mean ± SEM (3 experimental replicates, triangles, 2–5 technical replicates, circles). P values (Student’s two-tailed t test), **P ≤ 0.01. (E and F) PC3 acini expressing mNG, (E) ARF1-mNG, or (F) ARF3-mNG and Scr shRNA were classified into Round, Spindle, and Spread. n = 6 and 4 experimental replicates for ARF1-mNG and ARF3-mNG, respectively, each with 2–4 technical replicates/condition. (E) 5,005 (mNG), 1,938 (ARF1-mNG) and (F) 9,320 (mNG), 8,699 (ARF3-mNG) mNG-positive acini quantified in total. Heatmaps, Area is mean of Z-score normalized values (purple to yellow). P values, Student’s t test, Bonferroni adjustment, represented by size of bubble. Heatmaps, Round, Spindle, or Spread is Log2 fold change from control (mNG; blue to red). Proportion of control at each time is Z-score normalized (white to black). P values, CMH test, Bonferroni adjusted, represented by size of bubble. Dot indicates P value (Breslow–Day test, Bonferroni-adjusted) for consistent effect magnitude. (G) Schema, Class 1 ARFs share 100% identical core region but differ in seven amino acids (AA) in N and C termini. ARF chimeras with ARF3 N-terminal and ARF1 C-terminal (3N/1C) and ARF3 C-terminal and ARF1 N-terminal (1N/3C) created. (H) Western blot of PC3 cells expressing mNG, ARF1-mNG, ARF3-mNG, and ARF-mNG chimeras for mNG and GAPDH, as loading control. Panels shown are representative of 3 independent lysate preparations. (I) ARF-GTP pulldown and representative Western blot for mNG, GST, and GAPDH, as loading control for both. n = 3 independent lysate preparations and pulldowns. Graphs show mean GGA3 binding ± SEM normalized to ARF3. P values (Student’s two-tailed t test), *P ≤ 0.05. (J and K) PC3 cells expressing mNG, ARF3-mNG, or ARF-mNG chimeras plated in 3D invasion assay. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/2. Scale bars, 100 μm. Magnified images of boxed regions shown. White and black arrowheads, invasive chain or sheet, respectively. RWD at t = Max1/2, normalized to mNG shown in K. Data is mean ± SEM (3 experimental replicates, triangles, 2–5 technical replicates, circles). P values (Student’s two-tailed t test), ***P ≤ 0.001 and ****P ≤ 0.0001. (L) Schema, ARF3 expression levels affect mode of invasion. Source data are available for this figure: SourceData F4.

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