Transcription amplification of speckle-associated genes flanking Hsp70 gene locus at 37°C. (A and D) Probed gene locations (gray boxes, magenta gene names) relative to Hsp70 genes in BAC construct (A) in CHOK1 cells and at endogenous locus (D) in human HAP1 cells. (B and E) Representative images of smRNA FISH (red) signals for specific BAC transgene (B) or endogenous gene (E) showing nascent transcripts associated (top) versus nonassociated (bottom) with nuclear speckles (intensities scaled differently for top and bottom). White arrowheads point to BAC (B) or nascent transcripts (E); empty arrowheads point to nuclear speckle. Scale bars = 1 µm. (C and F) Boxplots showing nascent transcript levels for three (C) or four (F) genes flanking BAC HSPA1B transgene (C) or endogenous Hsp70 locus (F) as function of speckle association (A) or nonassociation (N). Mean (square inside box), median (line inside box), box (interquartile range), ends of error bars (upper and lower limit), × (top: 99%, bottom: 1%), – (top: maximum, bottom: minimum). Intensities are normalized by the mean intensity at speckle-associated loci: fold differences (×) of the mean (median) between A versus N (black). In C, number of cells (A/N) = 112/44, 143/37, and 185/39; P = 0.0024, 0.013, and 0.016 (paired Wilcoxon signed-rank test) for VARS, LSM2, and C6orf48 BAC transgenes. in D, number of cells (A/N) = 168/64, 144/47, 123/52, and 203/86; P = 2.0 × 10−6, 1.4 × 10−5, 0.23, and 1.5 × 10−11 (paired Wilcoxon signed-rank test) for endogenous MSH5, VARS, LSM2, and C6orf48 genes.
