(A) Effect of Thoc2 silencing on polysome-associated RNAs. ESCs were transfected with NT or Thoc2 siRNAs, and cells were harvested 96 hours after transfection. Polysome-associated RNAs were purified by sucrose-gradient ultracentrifugation. Relative abundance of mRNAs in total (Total) or polysome-associated (Fraction 7–12) RNAs was determined by RT-qPCRs, and plotted as mean ± SEM from three independent experiments. (B) Effect of Thoc2 or Thoc5 silencing on mRNA export. ESCs were transfected with NT, Thoc2, or Thoc5 siRNAs. RNA Fluorescence in situ hybridization (RNA-FISH) was carried out for Nanog, Sox2, Klf4, Oct4 and β-actin 96 hours after transfection. Red: fluorescence signals from the FISH probes. Blue: DAPI staining of the nuclei. (C) Ratio of the RNA FISH signals in the nucleus versus cytoplasm was quantified by Metamorph from at least 10 images from three independent experiments. Values are plotted as mean ± SEM. See also Figure S4.