TSA at 165 nM induces maximal acetylation of histone H3 and near-maximal acetylation of histone H4.

Immunoblot analysis of acetylated histones H3 and H4. WT zebrafish larvae at 48h.p.f. were incubated with TSA at various concentrations, DMSO, or no treatment, for 24hours. Lane 1 (8.25 nM TSA), 2 (16.5 nM TSA), 3 (41.25 nM TSA), 4 (82.5 nM TSA), 5 (165 nM TSA), 6 (330 nM TSA), 7 (825 nM TSA), 8 (0.5% DMSO), and 9 (no treatment). Total protein was extracted from each group of larvae at 72h.p.f., and analyzed by immunoblotting using the indicated antibodies. The intensity and area of each protein band was quantified by densitometric analysis. Each value represents the ratio of acetylated histone H3 (AcH3) to total histone H3, and acetylated histone H4 (AcH4) to H4, relative to that of control (no treatment).

TSA impairs growth and disrupts morphogenesis of exocrine pancreas in zebrafish larvae with hyperacetylation of nucleosomal histones.

WT zebrafish larvae were incubated in the absence or presence of 165 nM TSA (added at 48h.p.f.) for 24hours and then analyzed. (A) Exocrine pancreas (arrows) was analyzed by in situ hybridization using trypsin anti-sense riboprobes. Pancreatic acinar morphology by immunohistochemistry using anti-cadherin (Cad) antibodies, followed by transverse histological sectioning. e.p. exocrine pancreas; i, intestine. (B) The larvae were pulse-labeled with BrdU and analyzed by immunohistochemistry using anti-BrdU antibodies, followed by transverse histological sectioning. The number (#) of DAPI+ nuclei, the number (#) of BrdU+ nuclei, and the proportion of cells in S phase (% BrdU+ nuclei) were determined. Result is presented as the mean + s.d., and * indicates statistical significance, # trend of statistical significance. (C) Total protein was extracted and analyzed for acetylated and total histones H3 and H4 by immunoblotting. Anti-total histones H3 and H4 antibodies and anti-actin antibodies were used as internal controls.

TSA and ML-60218 synergistically inhibit expansion of exocrine pancreas in zebrafish by impeding cell cycle progression, with enhanced induction of histone acetylation and cyclin-dependent kinase inhibitors.

Starting at 48h.p.f., WT larvae were incubated in the presence of TSA, ML-60218, TSA + ML-60218, or control (DMSO or untreated) for 24hours and then analyzed. (A) Dorsal view of larvae with the exocrine pancreas (arrows) analyzed by whole mount in situ hybridization using anti-sense trypsin riboprobes. (B) Exocrine pancreatic epithelial cells in the S phase (upper panel) and morphometric analysis of cell growth (lower panel). The larvae were pulse-labeled with BrdU, processed for immunohistochemistry with anti-BrdU or anti-cadherin antibodies, followed by histological analysis. Each column indicates the mean proportion of BrdU+ nuclei or cell growth (area inµm² per cell) in the exocrine pancreatic epithelia. (C) Total protein was extracted from the larvae and analyzed by immunoblotting using anti-acetylated histones H3 and H4 antibodies, and anti-total histones H3 and H4 antibodies. (D) Total RNA was extracted from the larvae and quantified for p21^cdkn1a and p27^cdkn1b mRNA using real-time PCR. Each column represents the mean p21^cdkn1a or p27^cdkn1b mRNA level normalized to gapdh mRNA and expressed as percentage of control from three independent experiments, with each real-time PCR conducted in triplicate samples. Bars represent s.e.m.; *P<0.05 considered statistically significant; NS, not statistically significant.