3D visualization of an interconnected ANC channel network leading to nuclear pores in autosomal and Barr body regions. (A) 3D volume rendering of a DAPI-stained C2C12 nucleus imaged with 3D-SIM. Entire nuclear volume (left panels) and mid z-section (right panel) is shown. White boxes mark the regions shown in detail in (B,C,D). Scale bar: 5 μm. (B) Single z-section from autosomal (left) and Barr body region (right). Note that the autosomal chromatin is pervaded by an ANC network, occasionally forming large IC lacunae. This channel network is distinctly narrowed in the Barr body and lacks larger IC lacunae. Scale bar: 1 μm. (C) Left panel: 3D volume rendering of DAPI-stained chromatin (brown) and representation of the inverted DAPI signal within the nuclear interior marking the ANC compartment (green) of the two cuboids depicted in (A). Right panel: inverted DAPI signal only. Inset magnification shows the cropped Barr body with a rudimentary channel system representing the collapsed ANC. Scale bars: 1 μm, inset 0.5 μm. (D) Top view of the same region as in (C) with green channels leading to the nucleus’ surface in autosomal as well as in Barr body chromatin (left). Nuclear pore complex immunostaining with antibodies against Nup153 (blue) demonstrates the overlap of ANC channel signals (green) at the nuclear surface with nuclear pore complexes (right) (see also respective movies provided in Additional files 5, 6 and 7). Scale bar: 1 μm. 3D-SIM, three-dimensional structured illumination microscopy; ANC, active nuclear compartment; DAPI, 4',6-diamidino-2-phenylindole; IC, interchromatin compartment.