Details of the high throughput functional screen. (A) Validation of the colocalization analysis. MEF coilin knockout cell lines transiently expressing fluorescently tagged coilin (magenta) were immunostained with TMG (tri-methyguanosine cap) antibody (cyan). Colocalization analysis result is shown on the right. The total number of CBs analyzed (n) and the Pearson’s correlation coefficient (PCC) are listed. (B) Frequency histogram of Pearson’s correlation coefficient between nucleophosmin and coilin fluorescent signal per siRNA in screen. Values of siRNAs that relocalize coilin to nucleoli are highlighted in purple (Pearson’s > 0.75). (C) Representative images of hits showing relocalization of coilin to nucleoli (siMED14 and siUSPL1). Immunofluorescent images show α-coilin antibody in magenta and α-nucleophosmin antibody in cyan. Histograms for each hit quantifying the number of CBs per nucleus from their respective well and plate in the screen (12 fields of view). (D) Analysis of cell ploidy and CB integrity after knockdown of proteins that increase CB number. Representative histograms of Hoechst log₂ integrated intensity in samples treated with siNT or siRNAs targeting hits that increase CB numbers upon depletion normalized to siNT control. G0/G1 phase (0.75–1.25) highlighted in orange, S phase (1.25–1.75) in white/black font, G2/M phase (1.75–2.25) highlighted in purple, >4N (>2.5) highlighted in teal. On the right are cell cycle values showing % of total in each phase, quantified from values from the left.