Fig. 3

Fig. 3 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.