Fluorescent in situ hybridization (FISH) was carried out on cells that adhered to glass coverslips coated with ECM gel (Sigma-Aldrich) using nick-translated BAC DNA (BAC RP23-471-MyoD Locus) that incorporate Cy 3–deoxyuridine triphosphate (Enzo Life Sciences). Three-dimensional (3D) DNA FISH on FAPs was performed with some modifications of already described procedures (69 (link)). Briefly, cells were fixed in 4% PFA (20 min at 4°C) and preprocessed by freeze-thawing permeabilization to ensure the preservation of nuclear structures. After denaturation, FISH probes were hybridized overnight at 37°C, and samples were washed, DAPI-stained, and mounted in ProLong Diamond reagent (Thermo Fisher Scientific). Samples were imaged on inverted microscope (Olympus IX73) equipped with a confocal imager (Crest X-Light) spinning disk, a CoolSNAP MYO charge-coupled device camera (Photometrics), and a Lumencor Spectra X light-emitting diode illumination. Images were acquired with 60× NA 1.35 oil objective (UPlanSAPO) and MetaMorph (Molecular Devices) using 300 ms (Cy3), 300 ms (fluorescein), or 100 ms (DAPI) as exposure time. Confocal images were taken with a 0.2-μm-step Z-stacks. 3D reconstructions and 3D distance between the center of mass of the DNA FISH spots and the inner surface of the lamina were performed by using Huygens Professional software.