Expression levels of ARF GTPases vary in different prostate cancer cell lines in both 2D and 3D. (A–E) Graphs generated using RNA-seq data from the CCLE comparing (A) ARF1, (B) ARF3, (C) ARF4, (D) ARF5, and (E) ARF6 gene copy number and mRNA expression levels in multiple prostate cancer and non-transformed cell lines. Metastatic PC3 and normal prostate PLECLH cell lines, red and green, respectively. (F) Western blot of androgen receptor (AR) proficient or deficient prostate cell lines for ARF1, ARF3, ARF4, ARF5, and ARF6. GAPDH is loading control for ARF6 and a sample control for all other blots. Panels shown are representative of 3 independent lysate preparations. (G) Western blot of RWPE-1 and PC3 acini, formed in GFRM (3D) for 2 d, for ARF1, ARF3, ARF4, ARF5, and ARF6. GAPDH is loading control for ARF4 and a sample control for all other blots. Panels shown are representative of 3 independent lysate preparations. Graph is fold change of ARF expression in PC3 cells, normalized to RWPE-1 cells. Data presented as 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. (H and I) Western blot of PC3 cells in 2D or in 3D for (H) ARF1 or (I) ARF3. GAPDH is loading control for each ARF blot. Graph, fold change of ARF expression, normalized to 2D samples. Dashed lines indicate blot was spliced. Data presented as mean ± SEM and panels shown are representative of 3 independent lysate preparations. P values (Student’s two-tailed t test), **P ≤ 0.01, ***P ≤ 0.001. (J) RNA-seq data from PC3 cells shows mRNA expression (Log₂) of genes encoding ARF GTPases, GEFs, GAPs, components of the IL6 signaling pathway, and ARF effectors and interactors (Log₂). n = 4 mRNA samples prepared independently. (K) PC3 cells expressing ARFome shRNA were plated on ECM with 2% ECM overlay and multi-day high-throughput imaging carried out live in 3D. Mem:Venus-positive acini classified into Round, Spindle, or Spread phenotypes at each time point. Heatmap presents this classification, in 12-h time intervals, as a Log₂ fold change from control (Scr; blue to red). The proportion of control at each time point is Z-score normalized for each class (white to black). P values, CMH, Bonferroni adjusted, represented by the size of the bubble. Dot indicates P value (Breslow–Day test, Bonferroni-adjusted) for consistent effect magnitude. shRNAs grouped into seven groups (Phenotype Group 1–7) based on dendrogram generated using hierarchical clustering by complete linkage of Euclidian distances between samples. Viral infections and live 3D assays carried out 3 independent times. Each experimental replicate consisted of 18 technical replicates of Scr shRNA (170,674 acini in total) and 1 replicate of each of the 210 ARFome shRNAs (Table S1). (L and M) Western blot of PC3 cells expressing mNG and Scr, (L) ARF1, or (M) ARF3 shRNA for ARF1, ARF3, and GAPDH as a loading control for each. Panels shown are representative of 3 independent lysate preparations. Graph is fold change of ARF expression normalized to Scr. Data is mean ± SEM. P values (Student’s two-tailed t test), **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. Source data are available for this figure: SourceData FS1.