Figure 3.
Class I ARF GTPases regulate migration and invasion of prostate cancer cells. (A) Schema, 2D migration or 3D invasion (+ECM) of wounded PC3 monolayer. Three modes of movement observed, cells moving individually (I), as a sheet (S), or as chains (C). (B and C) Phase images of cells expressing mNG and Scr or (B) ARF1 or (C) ARF3 shRNA in 2D migration assay. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/2. Scale bars, 100 μm. Magnified images of boxed regions at different times shown. Graph is RWD at t = Max1/2 (Scr = 50% closed). Data is mean ± SEM (4 experimental replicates, triangles, 2–4 technical replicates, circles). P values (Student’s two-tailed t test), *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001. (D and E) Cells in a wounded monolayer overlaid with 25% ECM were imaged to observe 3D invasion. Phase images of cells expressing mNG and Scr, (D) ARF1 or (E) ARF3 shRNA shown. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/2. Scale bars, 100 μm. Magnified phase images of boxed regions at different times shown. White arrowheads, invasive chains. Graph is RWD at t = Max1/2, normalized to Scr. Data is mean ± SEM. (3–4 experimental replicates, triangles, 3–5 technical replicates, circles). P values (Student’s two-tailed t test), **P ≤ 0.01 and ****P ≤ 0.0001. (F) Western blot analysis of PC3 cells expressing Scr/Scr or ARF1/3_KD shRNA for ARF1 or ARF3. GAPDH is loading control for ARF3 and sample control for ARF1. Panels shown are representative of 3 independent lysate preparations. (G) Phase images of acini expressing Scr/Scr or ARF1/3_KD shRNA. Outlines: Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 5 experimental replicates each with 3–4 technical replicates/condition. 20,645 (Scr/Scr), 8,601 (ARF1/3) mem:Venus-positive acini quantified in total. (H) Quantitation of G. 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 (Scr/Scr; blue to red). Proportion of control at each time is also 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. (I) 2D PC3 cells expressing Scr/Scr or ARF1/3_KD shRNA classified into Round, Spindle, and Spread. Heatmaps, Log2 fold change over Scr/Scr. P values, one-way ANOVA, grayscale values as indicated. n = 2 experimental replicates with 4 technical replicates/condition. 3,323 (Scr/Scr) and 1,847 (ARF1/3) mem:Venus-positive cells quantified in total. (J) Phase images of cells expressing Scr/Scr or ARF1/3_KD shRNA in 3D invasion assay shown. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/2. Scale bars, 100 μm. Magnified image of boxed region shown. White arrowhead, invasive chain. Graph is RWD at t = Max1/2, normalized to Scr/Scr. Data is mean ± SEM (5 experimental replicates, triangles, 4–5 technical replicates, circles). P values (Student’s two-tailed t test), ****P ≤ 0.0001. Source data are available for this figure: SourceData F3.

Class I ARF GTPases regulate migration and invasion of prostate cancer cells. (A) Schema, 2D migration or 3D invasion (+ECM) of wounded PC3 monolayer. Three modes of movement observed, cells moving individually (I), as a sheet (S), or as chains (C). (B and C) Phase images of cells expressing mNG and Scr or (B) ARF1 or (C) ARF3 shRNA in 2D migration assay. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/2. Scale bars, 100 μm. Magnified images of boxed regions at different times shown. Graph is RWD at t = Max1/2 (Scr = 50% closed). Data is mean ± SEM (4 experimental replicates, triangles, 2–4 technical replicates, circles). P values (Student’s two-tailed t test), *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001. (D and E) Cells in a wounded monolayer overlaid with 25% ECM were imaged to observe 3D invasion. Phase images of cells expressing mNG and Scr, (D) ARF1 or (E) ARF3 shRNA shown. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/2. Scale bars, 100 μm. Magnified phase images of boxed regions at different times shown. White arrowheads, invasive chains. Graph is RWD at t = Max1/2, normalized to Scr. Data is mean ± SEM. (3–4 experimental replicates, triangles, 3–5 technical replicates, circles). P values (Student’s two-tailed t test), **P ≤ 0.01 and ****P ≤ 0.0001. (F) Western blot analysis of PC3 cells expressing Scr/Scr or ARF1/3_KD shRNA for ARF1 or ARF3. GAPDH is loading control for ARF3 and sample control for ARF1. Panels shown are representative of 3 independent lysate preparations. (G) Phase images of acini expressing Scr/Scr or ARF1/3_KD shRNA. Outlines: Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 5 experimental replicates each with 3–4 technical replicates/condition. 20,645 (Scr/Scr), 8,601 (ARF1/3) mem:Venus-positive acini quantified in total. (H) Quantitation of G. 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 (Scr/Scr; blue to red). Proportion of control at each time is also 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. (I) 2D PC3 cells expressing Scr/Scr or ARF1/3_KD shRNA classified into Round, Spindle, and Spread. Heatmaps, Log2 fold change over Scr/Scr. P values, one-way ANOVA, grayscale values as indicated. n = 2 experimental replicates with 4 technical replicates/condition. 3,323 (Scr/Scr) and 1,847 (ARF1/3) mem:Venus-positive cells quantified in total. (J) Phase images of cells expressing Scr/Scr or ARF1/3_KD shRNA in 3D invasion assay shown. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/2. Scale bars, 100 μm. Magnified image of boxed region shown. White arrowhead, invasive chain. Graph is RWD at t = Max1/2, normalized to Scr/Scr. Data is mean ± SEM (5 experimental replicates, triangles, 4–5 technical replicates, circles). P values (Student’s two-tailed t test), ****P ≤ 0.0001. Source data are available for this figure: SourceData F3.

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