Hsp70 transgenes associate with nuclear speckles after heat shock. (A) Hsp70 transcripts, detected by RNA FISH (red), accumulate within a DAPI (grayscale) poor region encircled by GFP-LacI signals (green) marking the Hsp70 transgene locations. (B and C) Close association of Hsp70 BAC transgenes, marked by EGFP-LacI (green), with nuclear speckles immunostained either with ASF/SF2 (B) or SC35 (C) antibodies (red) in Hsp70_5 cells 30 min after heat shock (bottom panel) as compared with nonheat shocked control cells (top panel). Grayscale shows DNA staining (DAPI). Bar (A–C), 2 µm. (D) In vivo immunogold staining of EGFP-LacI marks transgene location (arrows) associated with IGC (nuclear speckle). Enlarged area (right) shows that transgenes appear at the periphery of the dense cluster of granules defining the IGC (arrowheads). Bar, 1 µm. (E) Comparison of the diameter of the nearest nuclear speckle (average of longest and shortest speckle axes, green) versus transgene association index (red, see text) at different times after heat shock. Transgene association index increases from 50–90% during heat shock with no major change in average size of nearest speckle.