FIG 4.

S. granarius fibroblasts carry APB-like structures. (A) S. granarius primary fibroblasts (passage 7) were transfected with a plasmid expressing an ECFP-ICP0* fusion to reveal putative PML nuclear bodies. The native fluorescence of the ECFP-ICP0* fusion in three transfected nuclei is shown. ICP0* formed 7 to 24 (18 ± 1.94 [mean ± SEM]) large nuclear foci per transfected nucleus (n = 150 nuclei). (B) Cells expressing ECFP-ICP0* were costained with antibodies against the human shelterin protein RAP1 (green) and with a telomeric C-rich PNA probe (Cy3; red) and mounted in Vectashield with DAPI. Maximum-intensity projections are presented for all color channels. The arrows point to close contacts between RAP1, telomeres, and ICP0* (readily visible in the merged image on the far right), suggesting that they are part of the same APB-like structure. (C) 3D reconstitution of the images in panel B. (D) Immuno-FISH on S. granarius metaphase chromosomes, combining antibodies to human RAP1 (green) and a telomeric PNA probe (Cy3; red); chromosomes are stained with DAPI. Bars, 10 μm.