Figure 5.

A phenotypic screen for modifiers of disrupted nucleocytoplasmic transport in U-2 OS cells expressing PR₅₀. (A) Schematic of the phenotypic screen workflow. U-2 OS cells stably expressing NCT-C biosensor were plated 24 h prior to PR₅₀ transfection and treated with compounds at 3 µM 4 h thereafter. After 24 h of compound exposure, plates were fixed with 4% paraformaldehyde, stained with DAPI and an anti-PR antibody prior to image acquisition. (B) Scatterplot of compound Z-factors across all compound libraries at a single concentration (3 µM) in the presence of PR₅₀. A more negative Z-factor indicates restored NCT-C; whereas, a more positive Z-factor indicates disrupted NCT-C. (C) A confirmatory screen of the translocation index of top compounds from the primary screen mentioned above. Cells were either treated with vehicle (DMSO), untreated and expressing PR₅₀ (PR), or treated with compound at 3 µM for 24 h in the presence of PR₅₀. Each condition was repeated 4 times. (D) A scatterplot of cell counts under each condition from the confirmatory screen mentioned above. (E) A pie chart indicating the class of each hit compound tested in the confirmatory screen. * p < 0.05; *** p < 0.001; **** p < 0.0001; ns = non-significant.