Statistics for classifications of relative movements between BAC transgene and nuclear speckle. (A) Cartoons of representative dynamics (categories 1–3, top) during HS and corresponding averaged plots of live-cell data (bottom, mean ± SEM, n = 21–47 movies). Distance (d, green) of HSPA1B transgene from closest speckle and nascent RNA levels (red) in arbitrary units. Category 1 (left): stable speckle association of HSPA1B transgene and transcription increase after temperature reaches 42°C, 47 movies; category 2: transcription increase as function of time after transgene association with speckle (t = 0), 24 movies; category 3: transcription decrease as function of time after transgene dissociation (t = 0) from speckle, 21 movies. (B) Numbers for categories of dynamics observed for 20 to ∼25 min with or without HS. dmax, maximum distance between transgene locus and speckle during imaging; d1, distance between transgene locus and speckle at first time point; dn, distance between transgene locus and speckle at any time point during imaging; F, forward relative motion bringing transgene and speckle closer; B, backward relative motion moving transgene and speckle apart; +α, additional movements. (C) Three models for correlation of nascent transcript increase with speckle association: nuclear speckle (green), gene (yellow), and nascent transcripts (red; solid lines, intact; dashed lines, degraded). Left: Current working model. Nascent transcripts increase after speckle association through a decreased, exosome-mediated nascent transcript degradation rate, and possibly an increased transcription rate. Middle: Alternative model 1. Nascent transcripts adjacent to transcription site increase due to a reduced transcript release rate; at steady state, this would not change the rate of mRNA production and is inconsistent with the increased mRNA count 15 min after HS. Right: Alternative model 2. Nascent transcript levels increase before speckle association, causing subsequent speckle association; this is inconsistent with live-cell imaging temporal ordering of events.