Figure 5.
Identification of co-acting partnerships in the ARFome that regulate collective morphogenesis. (A) Western blot of PC3 cells expressing mNG and Scr or PSD shRNA for PSD, ARF6, ARF3, and actin or GAPDH, as loading control. Panels shown are representative of 3 independent lysate preparations. Quantitation of ARF6 and ARF3 expression is shown as mean fold change ± SEM normalized to Scr. P values (Student’s two-tailed t test), *P ≤ 0.05 and **P ≤ 0.01. (B) ARF-GTP pulldown and representative Western blot for ARF6, ARF3, and GST as loading control for ARFs in Scr or PSD shRNA cells. n = 5 independent lysate preparations and pulldowns. Graphs show mean GGA3 binding ± SEM normalized to ARF. P values (Student’s two-tailed t test), ****P ≤ 0.0001. (C) Phase images of PC3 acini expressing mNG and Scr or PSD shRNA. Outlines: Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 2 experimental replicates each with 4 technical replicates/condition. 10,723 (Scr), 13,920 (PSD_KD1), 15,157 (PSD_KD2) acini quantified in total. (D) Quantitation of C. 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; 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. (E) PC3 cells expressing mNG and Scr or PSD shRNA in 3D invasion assay. 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 chains. RWD at t = Max1/2 is shown, normalized to Scr. Data is mean ± SEM (3 experimental replicates, triangles, 4–8 technical replicates, circles). P values (Student’s two-tailed t test), *P ≤ 0.05 and ***P ≤ 0.001. (F) IP was performed using an anti-ARF3 antibody or mouse IgG and samples immunoblotted for RAB11FIP4 and ARF3. Panels shown are representative of 3 IPs from 3 independent lysate preparations. (G) IP was performed using mNG-Trap Agarose beads in cells expressing mNG or ARF3-mNG. Samples were immunoblotted for RAB11FIP4 and mNeonGreen. Panels shown are representative of 2 IPs from independent lysate preparations. (H) Image of PC3 cell stained with ARF3 (green) and RAB11FIP4 (red). Scale bars, 20 μm. Magnified images of boxed regions shown (a–c). Scale bars, 10 μm. Images representative of phenotypes observed in 3 experimental replicates. 40.2 ± 4% of ARF3 positive puncta overlap with RAB11FIP4 positive puncta in 320 cells quantified. (I and J) PC3 cells expressing mNG and Scr or ARF3 shRNA were stained for RAB11FIP4, High-Content Screening Whole Cell Stain (HCS WCS), and Hoechst. Number and area of RAB11FIP4 puncta was quantified (I) per cell or (J) per sub-cellular region in each cell. n = 3 independent experiments with 508 (Scr) and 613 (ARF3 KD) cells quantified in total. Data are presented as mean ± SEM normalized to Scr. P values (Student’s two-tailed t test). (K) Western blot of PC3 cells expressing mNG and Scr or RAB11FIP4 shRNA for RAB11FIP and GAPDH, as a loading control. Panels shown are representative of 3 independent lysate preparations. (L) Phase images of PC3 acini expressing mNG and Scr or RAB11FIP4 shRNA. Outlines: Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 3 experimental replicates each with 3–4 technical replicates/condition. 14,551 (Scr), 16,435 (RAB11FIP4_KD1), 11,880 (RAB11FIP4_KD2) acini quantified in total. (M) Quantitation of L. 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; 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. (N) PC3 cells expressing mNG and either Scr or RAB11FIP4 shRNA in 3D invasion assay. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/4. Scale bars, 100 μm. Magnified image of boxed region shown. White arrowhead, invasive chains. RWD at t = Max1/4 is shown, normalized to Scr. Data is mean ± SEM. (3 experimental replicates, triangles, 3–8 technical replicates, circles). P values (Student’s two-tailed t test), ****P ≤ 0.0001. (O) Schema, relationship between PSD, ARF3, and Rab11FIP4. Source data are available for this figure: SourceData F5.

Identification of co-acting partnerships in the ARFome that regulate collective morphogenesis. (A) Western blot of PC3 cells expressing mNG and Scr or PSD shRNA for PSD, ARF6, ARF3, and actin or GAPDH, as loading control. Panels shown are representative of 3 independent lysate preparations. Quantitation of ARF6 and ARF3 expression is shown as mean fold change ± SEM normalized to Scr. P values (Student’s two-tailed t test), *P ≤ 0.05 and **P ≤ 0.01. (B) ARF-GTP pulldown and representative Western blot for ARF6, ARF3, and GST as loading control for ARFs in Scr or PSD shRNA cells. n = 5 independent lysate preparations and pulldowns. Graphs show mean GGA3 binding ± SEM normalized to ARF. P values (Student’s two-tailed t test), ****P ≤ 0.0001. (C) Phase images of PC3 acini expressing mNG and Scr or PSD shRNA. Outlines: Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 2 experimental replicates each with 4 technical replicates/condition. 10,723 (Scr), 13,920 (PSD_KD1), 15,157 (PSD_KD2) acini quantified in total. (D) Quantitation of C. 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; 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. (E) PC3 cells expressing mNG and Scr or PSD shRNA in 3D invasion assay. 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 chains. RWD at t = Max1/2 is shown, normalized to Scr. Data is mean ± SEM (3 experimental replicates, triangles, 4–8 technical replicates, circles). P values (Student’s two-tailed t test), *P ≤ 0.05 and ***P ≤ 0.001. (F) IP was performed using an anti-ARF3 antibody or mouse IgG and samples immunoblotted for RAB11FIP4 and ARF3. Panels shown are representative of 3 IPs from 3 independent lysate preparations. (G) IP was performed using mNG-Trap Agarose beads in cells expressing mNG or ARF3-mNG. Samples were immunoblotted for RAB11FIP4 and mNeonGreen. Panels shown are representative of 2 IPs from independent lysate preparations. (H) Image of PC3 cell stained with ARF3 (green) and RAB11FIP4 (red). Scale bars, 20 μm. Magnified images of boxed regions shown (a–c). Scale bars, 10 μm. Images representative of phenotypes observed in 3 experimental replicates. 40.2 ± 4% of ARF3 positive puncta overlap with RAB11FIP4 positive puncta in 320 cells quantified. (I and J) PC3 cells expressing mNG and Scr or ARF3 shRNA were stained for RAB11FIP4, High-Content Screening Whole Cell Stain (HCS WCS), and Hoechst. Number and area of RAB11FIP4 puncta was quantified (I) per cell or (J) per sub-cellular region in each cell. n = 3 independent experiments with 508 (Scr) and 613 (ARF3 KD) cells quantified in total. Data are presented as mean ± SEM normalized to Scr. P values (Student’s two-tailed t test). (K) Western blot of PC3 cells expressing mNG and Scr or RAB11FIP4 shRNA for RAB11FIP and GAPDH, as a loading control. Panels shown are representative of 3 independent lysate preparations. (L) Phase images of PC3 acini expressing mNG and Scr or RAB11FIP4 shRNA. Outlines: Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 3 experimental replicates each with 3–4 technical replicates/condition. 14,551 (Scr), 16,435 (RAB11FIP4_KD1), 11,880 (RAB11FIP4_KD2) acini quantified in total. (M) Quantitation of L. 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; 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. (N) PC3 cells expressing mNG and either Scr or RAB11FIP4 shRNA in 3D invasion assay. Yellow lines, initial wound, and red pseudo color, wound at t = Max1/4. Scale bars, 100 μm. Magnified image of boxed region shown. White arrowhead, invasive chains. RWD at t = Max1/4 is shown, normalized to Scr. Data is mean ± SEM. (3 experimental replicates, triangles, 3–8 technical replicates, circles). P values (Student’s two-tailed t test), ****P ≤ 0.0001. (O) Schema, relationship between PSD, ARF3, and Rab11FIP4. Source data are available for this figure: SourceData F5.

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