Identify proteins required for paraspeckle formation using shRNA screen. (A and B) Representative images showed colocalization of newly identified paraspeckle proteins with paraspeckles marker p54nrb (A) or NEAT1 long noncoding RNA (B). (C–E) Phenotypic screen for proteins affecting paraspeckles assembly. (C) A schematic flow for shRNA screen is presented. (D) Bar graph showed the percentage (±SD) of paraspeckles-containing cells determined by p54nrb or NEAT1 staining after the indicated shRNA treatment relative to control mock shRNA-treated cells (n = 3 independent experiments). (E) Bar graph showed relative NEAT1 expression (±SD) to control mock shRNA-treated cells and normalized to GAPDH (n = 3 independent experiments). Dotted lines display the level of control mock shRNA-treated cells for comparison. (F) Representative images showed phenotypes after shRNA transduction. The localization of paraspeckles foci was detected with the use of anti-p54nrb antibodies (top) or FITC-RNA probes against NEAT1 (bottom). Arrows show the paraspeckles foci labeled by anti-p54nrb antibodies (top) or RNA probes against NEAT1 (bottom). CTL, control. Bars, 10 µm.