Figure 2.
Contribution of each component of the ARFome to collective cancer cell behavior was examined by individual depletion. (A) Schema, PC3 acini expressing mem:Venus shRNAs were imaged, tracked, and classified. Phenotype Group 1–7 identified based on frequency of classification into Round, Spindle, and Spread over time. Interaction map shown, shRNAs color-coded by Phenotype Group. (B) Graphs show percentage of acini classified as Round (red), Spindle (green), and Spread (blue) for each Phenotype Group and Scr shRNA (control, on each graph). Data are mean, shaded regions represent SEM. Viral infections and live 3D spheroid assays carried out 3 independent times. Each experimental replicate consisted of 18 technical replicates of Scr (170,674 acini in total) and 1 replicate of 210 ARFome shRNAs (Table S1). (C) STRING network analysis of acini visualized using Cytoscape. Phenotype Groups 1–7 identified by frequency of acini classification into Round, Spindle, and Spread. Colors indicate Phenotype Group, and the proportion of shRNAs for each target that sit in different groups is shown. (D) Graph is mean percentage of acini, across all time points, classified into Round, Spindle, and Spread per Phenotype Group. (E) PC3 acini expressing mNG and Scr, ARF1, or ARF3 shRNA. Outlines: Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 4 and 6 experimental replicates for ARF1 and ARF3 shRNA, respectively, each with 4 technical replicates/condition. 16,760 (Scr), 21,086 (ARF1_KD2), 19,424 (ARF1_KD4), and 31,414 (Scr), 40,135 (ARF3_KD1), 30,460 (ARF3_KD2) acini quantified in total. (F and G) Quantitation of E. 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.

Contribution of each component of the ARFome to collective cancer cell behavior was examined by individual depletion. (A) Schema, PC3 acini expressing mem:Venus shRNAs were imaged, tracked, and classified. Phenotype Group 1–7 identified based on frequency of classification into Round, Spindle, and Spread over time. Interaction map shown, shRNAs color-coded by Phenotype Group. (B) Graphs show percentage of acini classified as Round (red), Spindle (green), and Spread (blue) for each Phenotype Group and Scr shRNA (control, on each graph). Data are mean, shaded regions represent SEM. Viral infections and live 3D spheroid assays carried out 3 independent times. Each experimental replicate consisted of 18 technical replicates of Scr (170,674 acini in total) and 1 replicate of 210 ARFome shRNAs (Table S1). (C) STRING network analysis of acini visualized using Cytoscape. Phenotype Groups 1–7 identified by frequency of acini classification into Round, Spindle, and Spread. Colors indicate Phenotype Group, and the proportion of shRNAs for each target that sit in different groups is shown. (D) Graph is mean percentage of acini, across all time points, classified into Round, Spindle, and Spread per Phenotype Group. (E) PC3 acini expressing mNG and Scr, ARF1, or ARF3 shRNA. Outlines: Round (red), Spindle (green), and Spread (blue). Scale bar, 100 μm. n = 4 and 6 experimental replicates for ARF1 and ARF3 shRNA, respectively, each with 4 technical replicates/condition. 16,760 (Scr), 21,086 (ARF1_KD2), 19,424 (ARF1_KD4), and 31,414 (Scr), 40,135 (ARF3_KD1), 30,460 (ARF3_KD2) acini quantified in total. (F and G) Quantitation of E. 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.

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