Figure S3.
ARF1 or ARF3 over-expression has different effects on migration and invasion of prostate cancer cells. (A–D) Phase images of cells expressing mNG, (A) ARF1-mNG or (C) ARF3-mNG and Scr 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 shown. RWD at t = Max1/2, normalized to mNG is shown for (B) ARF1-mNG or (D) ARF3-mNG. Data is mean ± SEM (4–5 experimental replicates, triangles, 3–4 technical replicates, circles). P values (Student’s two-tailed t test), ***P ≤ 0.001. (E) Confocal images of PC3 cells expressing mNG, ARF3-mNG or ARF-mNG chimeras (black, inverted images). Scale bars, 20 μm. Magnified images of boxed regions shown (a–h). Scale bars, 10 μm. White arrows, ARF mNG in discrete puncta, black arrows, areas of concentrated peri-nuclear staining. n = 3 experimental replicates. (F) Quantitation of E (upper panels). n = 3 experimental replicates with 43 (ARF3), 97 (3N/1C), 76 (1N/3C) cells quantified in total. Graph is percentage of cells with mNG concentrated in the peri-nuclear region. Data is mean ± SEM. P values (Student’s two-tailed t test), ***P ≤ 0.001. (G) 2D PC3 cells expressing mNG, ARF3-mNG, or ARF-mNG chimeras classified into Round, Spindle, and Spread. Heatmaps, Log2 fold change over mNG. P values, one-way ANOVA, grayscale values as indicated. n = 2 experimental replicates with 4 technical replicates/condition. 4,746 (mNG), 7,342 (ARF3-mNG), 5,567 (3N/1C), and 4,717 (1N/3C) mNG-positive cells quantified in total. (H) Confocal images of PC3 acini stained with F-actin (red) and Hoechst (nuclei, blue; middle panels). Intensity of F-actin staining can be appreciated using FIRE LUT. Scale bars, 20 μm. n = 3 experimental replicates. (I) Maximum intensity projections of PC3 acini expressing ARF3-mNG or ARF-mNG chimeras (green) stained with GM130 or RAB11 (red). Scale bars, 20 μm. Magnified images of boxed regions are shown, single plane. Scale bars, 10 μm. n = 2 experimental replicates. (J) Phase images of PC3 acini (lower panels); Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 3 experimental replicates each with 3 technical replicates/condition. (K) Quantitation of J. n = 3 experimental replicates each with 3 technical replicates/condition. 3,765 (mNG), 2,218 (ARF3-mNG), 1,150 (3N/1C), and 3,067 (1N/3C) 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 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.

ARF1 or ARF3 over-expression has different effects on migration and invasion of prostate cancer cells. (A–D) Phase images of cells expressing mNG, (A) ARF1-mNG or (C) ARF3-mNG and Scr 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 shown. RWD at t = Max1/2, normalized to mNG is shown for (B) ARF1-mNG or (D) ARF3-mNG. Data is mean ± SEM (4–5 experimental replicates, triangles, 3–4 technical replicates, circles). P values (Student’s two-tailed t test), ***P ≤ 0.001. (E) Confocal images of PC3 cells expressing mNG, ARF3-mNG or ARF-mNG chimeras (black, inverted images). Scale bars, 20 μm. Magnified images of boxed regions shown (a–h). Scale bars, 10 μm. White arrows, ARF mNG in discrete puncta, black arrows, areas of concentrated peri-nuclear staining. n = 3 experimental replicates. (F) Quantitation of E (upper panels). n = 3 experimental replicates with 43 (ARF3), 97 (3N/1C), 76 (1N/3C) cells quantified in total. Graph is percentage of cells with mNG concentrated in the peri-nuclear region. Data is mean ± SEM. P values (Student’s two-tailed t test), ***P ≤ 0.001. (G) 2D PC3 cells expressing mNG, ARF3-mNG, or ARF-mNG chimeras classified into Round, Spindle, and Spread. Heatmaps, Log2 fold change over mNG. P values, one-way ANOVA, grayscale values as indicated. n = 2 experimental replicates with 4 technical replicates/condition. 4,746 (mNG), 7,342 (ARF3-mNG), 5,567 (3N/1C), and 4,717 (1N/3C) mNG-positive cells quantified in total. (H) Confocal images of PC3 acini stained with F-actin (red) and Hoechst (nuclei, blue; middle panels). Intensity of F-actin staining can be appreciated using FIRE LUT. Scale bars, 20 μm. n = 3 experimental replicates. (I) Maximum intensity projections of PC3 acini expressing ARF3-mNG or ARF-mNG chimeras (green) stained with GM130 or RAB11 (red). Scale bars, 20 μm. Magnified images of boxed regions are shown, single plane. Scale bars, 10 μm. n = 2 experimental replicates. (J) Phase images of PC3 acini (lower panels); Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 3 experimental replicates each with 3 technical replicates/condition. (K) Quantitation of J. n = 3 experimental replicates each with 3 technical replicates/condition. 3,765 (mNG), 2,218 (ARF3-mNG), 1,150 (3N/1C), and 3,067 (1N/3C) 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 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.

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