Figure 3

Localization of the episome in interphase nuclei. The qualitative co-localization of the episome with subnuclear structures in interphase nuclei was analyzed using pEPI FISH (A-J). In some experiments pEPI FISH was used in combination with immunofluorescence techniques to analyse the co-localization of pEPI with subnuclear marker proteins (E-J). Co-localizing voxels of two channels being analyzed were highlighted in white color in (A, B, C, D, H). The episome (green) was visualized by pEPI FISH. To-Pro-3 was used for DNA counterstaining (red) in (A, B, C, D, E, G, I). Maximum intensity projections were rendered from a set of 5 mid serial sections (A, B, C, D, E, G, H, I). For further details see Additional file 2. (A, B) Co-localization analyses showed that cells transfected with the linearized vectors displayed FISH signals consistent with integration of the vector at a single chromosomal locus as the vectors completely co-localized with domains containing condensed chromatin. Equal results were obtained when hyper-condensation of chromatin was induced prior to fixation (B). (C, D) Analyses of single pEPI signals revealed that the vector did not (green) or at most incompletely (white/green) colocalize to condensed chromatin regions (red). Insets are 400% magnifications of white or blue framed sectors in C and D. Such representative pEPI signals consist of green voxels (negative co-localization with condensed chromatin) or are composed of white and green voxels (incomplete co-localization with condensed chromatin). Negative or incomplete co-localization suggests that the episome is localized within the IC or at perichromatin regions bordering the IC at condensed chromatin surfaces. Equal results were obtained when hyper-condensation of chromatin was induced prior to fixation (B). (For selected light optical sections corresponding to the nuclei displayed in C and D as maximum intensity projections see the Additional file 2.) (E, F) Co-localization analyses of the episome (green) and SC35 (blue) showed that the episome occured co-localized or in close proximity to nuclear speckles, a structure which is found within the IC. (F) A 3D reconstruction was rendered from the same nucleus as displayed in (E). (G, H) Nuclear localization of the episome (green) and histone3 acetylated at lysines 9/14 (H3 acetyl-K9/K14) (blue). The nuclear counterstain (red) channel was hidden in (H) to facilitate co-localization analysis of the episome and H3 acetyl-K9/K14 showing that the episome occurred co-localized or in close proximity to sites of active transcription. (I, J) Co-localization analyses of the episome (green) and histone3 trimethylated at lysine9 (H3 trimethyl-K9) (blue) showed that the episome did not co-localize with such sites. (J) A 3D reconstruction was rendered from the same nucleus as displayed in (I). Bar in (A) is representative for (A-J).