Figure 1.
Figure 1. Both HSPA1B BAC transgenes and endogenous genes induce synchronously 2–4 min after HS but show higher transcript levels when associated with nuclear speckles. (A) Transcriptional induction of HSPA1B BAC transgene in CHOK1 cells and endogenous Hsp70 genes in HAP1 cells occurs 0–4 min after HS (SEM, three replicates). n = 110–210 for HAP1, n = 95–150 for CHOK1 cells, each replicate time point. (B) Scatterplot between levels of nascent RNA signals (RNA FISH intensity at HSPA1B locus) versus numbers of mature mRNAs after 15-min HS. n = 99 and 71 for A and N. P value (A versus N) = 8.5 × 10−7 for transcript signal, 1 × 10−6 for mRNA count; paired Wilcoxon signed rank test. rA, Pearson correlation coefficient for speckle-associated locus; rN, for nonassociated locus. (C) smRNA FISH image after 15-min HS. Nascent pre-mRNAs (top) at HSPA1B BAC transgene (arrowhead, middle). White dashes outline cell border. The boxed region around BAC is enlarged on left. (D) Higher pre-mRNA levels (boxed regions) for speckle-associated (top) versus nonassociated (bottom) BAC transgenes. (Left) Representative images of smRNA FISH versus nuclear speckles and BAC transgene at 0, 1, and 2 h after HS. (Right) Normalized pre-mRNA intensities at speckle-associated (black, A) or nonassociated (red, N) BAC transgenes 0, 1, and 2 h after HS, with fold differences (blue) of mean (median) for A versus N. Nascent RNA intensities were normalized by mean intensity of A at 2 h. Cell numbers (A/N) = 101/101, 91/40, and 107/63 at 0, 1, and 2 h. P = 4.5 × 10−11 at 1-h HS and 7.8 × 10−12 at 2-h HS for A versus N; 9.7 × 10−4 for A at 1 versus 2 h; and 8.4 × 10−4 for N at 1 versus 2 h; paired Wilcoxon signed-rank test. (E) Left: Same as in D but for endogenous Hsp70 locus in CHOK1 cells (left two panels) versus haploid human HAP1 cells (right two panels). Nonlinear intensity scaling used for red channel. Right: Same as in D, right, for endogenous Hsp70 locus in CHOK1 (left) versus HAP1 cells (right). Nascent RNA intensities were normalized by mean intensity of A at 2 h in CHOK1 cells and HAP1 cells, respectively. CHOK1: cell numbers (A/N) = 46/33 and 50/28 at 1 and 2 h, P = 1.8 × 10−5 at 1-h HS and 7.5 × 10−8 at 2-h HS for A versus N, 0.0038 for A at 1 versus 2 h, 0.0015 for N at 1 versus 2 h; HAP1: cell numbers (A/N) =102/44 and 112/33 at 1 and 2 h, P = 2.0 × 10−9 at 1-h HS and 6.5 × 10−9 at 2-h HS for A versus N, 4.6 × 10−4 for A at 1 versus 2 h, 0.12 for N at 1 versus 2 h; paired Wilcoxon signed-rank test.

Both HSPA1B BAC transgenes and endogenous genes induce synchronously 2–4 min after HS but show higher transcript levels when associated with nuclear speckles. (A) Transcriptional induction of HSPA1B BAC transgene in CHOK1 cells and endogenous Hsp70 genes in HAP1 cells occurs 0–4 min after HS (SEM, three replicates). n = 110–210 for HAP1, n = 95–150 for CHOK1 cells, each replicate time point. (B) Scatterplot between levels of nascent RNA signals (RNA FISH intensity at HSPA1B locus) versus numbers of mature mRNAs after 15-min HS. n = 99 and 71 for A and N. P value (A versus N) = 8.5 × 10−7 for transcript signal, 1 × 10−6 for mRNA count; paired Wilcoxon signed rank test. rA, Pearson correlation coefficient for speckle-associated locus; rN, for nonassociated locus. (C) smRNA FISH image after 15-min HS. Nascent pre-mRNAs (top) at HSPA1B BAC transgene (arrowhead, middle). White dashes outline cell border. The boxed region around BAC is enlarged on left. (D) Higher pre-mRNA levels (boxed regions) for speckle-associated (top) versus nonassociated (bottom) BAC transgenes. (Left) Representative images of smRNA FISH versus nuclear speckles and BAC transgene at 0, 1, and 2 h after HS. (Right) Normalized pre-mRNA intensities at speckle-associated (black, A) or nonassociated (red, N) BAC transgenes 0, 1, and 2 h after HS, with fold differences (blue) of mean (median) for A versus N. Nascent RNA intensities were normalized by mean intensity of A at 2 h. Cell numbers (A/N) = 101/101, 91/40, and 107/63 at 0, 1, and 2 h. P = 4.5 × 10−11 at 1-h HS and 7.8 × 10−12 at 2-h HS for A versus N; 9.7 × 10−4 for A at 1 versus 2 h; and 8.4 × 10−4 for N at 1 versus 2 h; paired Wilcoxon signed-rank test. (E) Left: Same as in D but for endogenous Hsp70 locus in CHOK1 cells (left two panels) versus haploid human HAP1 cells (right two panels). Nonlinear intensity scaling used for red channel. Right: Same as in D, right, for endogenous Hsp70 locus in CHOK1 (left) versus HAP1 cells (right). Nascent RNA intensities were normalized by mean intensity of A at 2 h in CHOK1 cells and HAP1 cells, respectively. CHOK1: cell numbers (A/N) = 46/33 and 50/28 at 1 and 2 h, P = 1.8 × 10−5 at 1-h HS and 7.5 × 10−8 at 2-h HS for A versus N, 0.0038 for A at 1 versus 2 h, 0.0015 for N at 1 versus 2 h; HAP1: cell numbers (A/N) =102/44 and 112/33 at 1 and 2 h, P = 2.0 × 10−9 at 1-h HS and 6.5 × 10−9 at 2-h HS for A versus N, 4.6 × 10−4 for A at 1 versus 2 h, 0.12 for N at 1 versus 2 h; paired Wilcoxon signed-rank test.

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