Z stack images of transfected and stained CECs were processed using Zeiss software. Images for each independent CEC line were taken using the same image acquisition parameters. Post capture, z stacks were processed as maximum intensity projection images. RNA foci were quantified by segmentation using CellProfiler (Broad Institute).28 Nuclei were defined in the 405 nm (DAPI) channel, after a median filter was applied, by adaptive maximum correlation thresholding, followed by form-factor and eccentricity filtering. RNA foci were defined in the 561 nm (Cy3) channel, after a median filter was applied, as objects within the perimeter of a nucleus, using per-nucleus robust background thresholding, with parameters set per cell line, due to differences in staining intensity. Cells with high background in FISH staining, defined as mean intensity per nucleus, were discarded to reduce segmentation error. A minimum of 100 nuclei per independent condition and cell line were analyzed.