Fig. 3.

A transcription wave visualized by RNA FISH. HUVECs were fixed at 0, 30, 52.5 and 75 min after stimulation with TNFα, and nascent SAMD4A or EDN1 RNA were detected by FISH using intron probes 1a, 1b, or 7, or an intron probe against EDN1 (labeled with Alexa 488), cells counterstained with DAPI, and images were collected. SAMD4 intron 1a (green), 1b (red), and 7 (blue) peaked as a wave of transcription passed through each region with time, whereas EDN1 signal (gray) remains constant (D, upper graph). Similar variations were given by relevant probes in microarrays (D, lower graph). (A) SAMD4A locus showing probe positions. (B) A typical field 30 min after stimulation obtained using probe 1a. Cells have 0, 1, or 2 green foci/cell (arrows) marking nascent RNA at one or other allele. Intensities are normalized relative to fluorescent beads (inset) to permit comparison between different experiments. Bar, 5 μm. (C) A nucleus 150 min after stimulation using probes 1a and 7; it contains one red and one green focus marking nascent RNA from each intron; yellow foci are never seen. (Inset) Positive control showing yellow focus given by probes 1a (green) and 1a-1 (red) 30 min after induction; these probes target intronic RNA sequences lying 1,000 nucleotides apart. Bar, 5 μm. (D) RNA FISH and arrays give similar results. (Top) Signals (i.e., size in pixels × intensity × number of foci; in arbitrary units [au]) were obtained by single (open symbols; as in B) or double labeling (closed symbols, as in C). (Bottom) Similar variations are given by relevant probes in arrays.