Fig. 3

Detection of HSat1A transcripts by RNA-FISH and RNA-FISH/IF. A HSat1A RNA-FISH with RNase A treatment. HSat1A transcripts were detected by RNA-FISH (red) in control and treated cells. Signal decrease in RNase-treated cells demonstrates that the observed signals are RNA-specific. Evaluation of the average intensity of active signal objects (all slices) in RNA-FISH control and RNA-FISH + RNase A was performed in “Counting and Tracking” (AutoQuant X3). Analysis shown in H1299. Values are mean ± SD (n = 20). ****p ≤ 0.0001 (unpaired t test). B Nuclear organization of HSat1A transcripts (red). Different spatial distribution and number of foci are observable between cell lines with similar amounts of HSat1A transcripts (RT-qPCR data). C Spatial organization of HSat1A transcripts in relation to nucleoli. HSat1A RNA-FISH (red) coupled with IF for fibrillarin detection (green). HSat1A transcripts seem to accumulate adjacently to nucleoli, as seen by confocal 3D image analysis for H1299 and MCF10A cells. Orthogonal slices for axis projections are displayed with isosurfaces for both channels. D HSat1A RNA-FISH (red) followed by HSat1A DNA-FISH (green). Merged confocal images show distinct signal features, with some co-localized signals. DNA is in blue (DAPI) in all the presented images. Scale bars represent 10 μm