IF experiments were carried out on cells grown on coverslips. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Coverslips were incubated with primary antibodies in 3% BSA for 1 h at room temperature. Coverslips were washed three times with PBS and incubated with secondary conjugated antibodies for 45 min. Coverslips were wash 3 times in PBS and fixed again before doing RNA FISH experiments. SmiFISH method was done as previously described (44 (link)) using Cy3-labeled DNA fragments specific to U3 and U85 snoRNAs. After incubation with the hybridization mix for one night, coverslips were all washed twice in 10% formamide in 2XSSC, once in PBS and mounted in Vectashield solution containing DAPI (Vector Laboratories). Samples were observed at RT using an upright epifluorescence microscope (LEICA DM6000) with a ×63 oil objective (NA 1.3). Images were captured with a CCD camera (Coolsnap HQ2 from Photometrics) using MetaMorph (Molecular Devices) and processed with Photoshop (Adobe). Deconvolution was proceeded with Huygens Professional (Scientific Volume Imaging).