Figure S4.
N-cadherin depletion mimics ARF3 depletion in 2D and 3D assays. (A) Quantitation of total F-actin intensity/acini described in Fig. 6 A. Box and whiskers plot, 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n = 2 experimental replicates with 643 (mNG, Scr shRNA), 712 (mNG, ARF3 KD1 shRNA), and 383 (ARF3-mNG, Scr shRNA) cells quantified in total. P values (Student’s two-tailed t test), ****P ≤ 0.0001. (B) Quantitation of percentage of PC3 acini described in Fig. 6 A with F-actin intensity visibly reduced in junctions. Data is mean ± SEM. n = 3 experimental replicates with 121 (mNG, Scr shRNA), 108 (mNG, ARF3 KD1 shRNA), and 121 (ARF3-mNG, Scr shRNA) cells quantified in total. P values (Student’s two-tailed t test), ***P ≤ 0.001. (C–F) Representative Western blots of PC3 cells expressing (C) mNG or (D) ARF3-mNG and Scr or N-cadherin shRNA for N-cadherin, E-cadherin, and ARF3 antibodies. GAPDH is a loading control for both cadherin blots and a sample control for ARF3. Graphs are fold change, normalized to Scr. Data is mean ± SEM for n = 3 or 5 independent lysate preparations for mNG (C and E) and 3 independent preparations for ARF3-mNG (D and F). P values (Student’s two-tailed t test), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. (G) 2D PC3 cells expressing mNG or ARF3-mNG and Scr or N-cadherin shRNA were classified into Round, Spindle, and Spread. Heatmaps, Log2 fold change over Scr. P values, one-way ANOVA, grayscale values as indicated. n = 3 experimental replicates with 3 technical replicates/condition. 19,184 (Scr), 37,230 (N-cadherin_KD1), 26,284 (N-cadherin_KD2) and 16,210 (Scr), 29,065 (N-cadherin_KD1), 46,352 (N-cadherin_KD2) cells were quantified for mNG or ARF3-mNG, respectively. (H) Representative phase images of PC3 acini expressing mNG and Scr or N-cadherin shRNA. Outlines: Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 5 experimental replicates each with 3–4 technical replicates/condition. 23,538 (Scr), 34,624 (N-cadherin_KD1), and 36,432 (N-cadherin_KD2) acini quantified in total. (I) Quantitation of H. 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. (J) Western blot of PC3 cells expressing mNG and Scr or RAB11FIP4 shRNA for N-cadherin and GAPDH, as a loading control. Graph is fold change, normalized to Scr. Data is mean ± SEM. Panels shown are representative of 3 independent lysate preparations. P values (Student’s two-tailed t test), *P ≤ 0.05 and **P ≤ 0.01. Source data are available for this figure: SourceData FS4.

N-cadherin depletion mimics ARF3 depletion in 2D and 3D assays. (A) Quantitation of total F-actin intensity/acini described in Fig. 6 A. Box and whiskers plot, 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n = 2 experimental replicates with 643 (mNG, Scr shRNA), 712 (mNG, ARF3 KD1 shRNA), and 383 (ARF3-mNG, Scr shRNA) cells quantified in total. P values (Student’s two-tailed t test), ****P ≤ 0.0001. (B) Quantitation of percentage of PC3 acini described in Fig. 6 A with F-actin intensity visibly reduced in junctions. Data is mean ± SEM. n = 3 experimental replicates with 121 (mNG, Scr shRNA), 108 (mNG, ARF3 KD1 shRNA), and 121 (ARF3-mNG, Scr shRNA) cells quantified in total. P values (Student’s two-tailed t test), ***P ≤ 0.001. (C–F) Representative Western blots of PC3 cells expressing (C) mNG or (D) ARF3-mNG and Scr or N-cadherin shRNA for N-cadherin, E-cadherin, and ARF3 antibodies. GAPDH is a loading control for both cadherin blots and a sample control for ARF3. Graphs are fold change, normalized to Scr. Data is mean ± SEM for n = 3 or 5 independent lysate preparations for mNG (C and E) and 3 independent preparations for ARF3-mNG (D and F). P values (Student’s two-tailed t test), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. (G) 2D PC3 cells expressing mNG or ARF3-mNG and Scr or N-cadherin shRNA were classified into Round, Spindle, and Spread. Heatmaps, Log2 fold change over Scr. P values, one-way ANOVA, grayscale values as indicated. n = 3 experimental replicates with 3 technical replicates/condition. 19,184 (Scr), 37,230 (N-cadherin_KD1), 26,284 (N-cadherin_KD2) and 16,210 (Scr), 29,065 (N-cadherin_KD1), 46,352 (N-cadherin_KD2) cells were quantified for mNG or ARF3-mNG, respectively. (H) Representative phase images of PC3 acini expressing mNG and Scr or N-cadherin shRNA. Outlines: Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 5 experimental replicates each with 3–4 technical replicates/condition. 23,538 (Scr), 34,624 (N-cadherin_KD1), and 36,432 (N-cadherin_KD2) acini quantified in total. (I) Quantitation of H. 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. (J) Western blot of PC3 cells expressing mNG and Scr or RAB11FIP4 shRNA for N-cadherin and GAPDH, as a loading control. Graph is fold change, normalized to Scr. Data is mean ± SEM. Panels shown are representative of 3 independent lysate preparations. P values (Student’s two-tailed t test), *P ≤ 0.05 and **P ≤ 0.01. Source data are available for this figure: SourceData FS4.

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