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Fig. 5
HDAC inhibitors inhibit neuromast cell proliferation. (a) Confocal images of neuromasts from a 5dpf control and from 5dpf embryos treated with 0.1μM TSA, 100μM VPA or 10μM MS-275 that have been labeled by BrdU to detect proliferating cells. The number of BrdU-labeled cells is much larger in control than in HDAC inhibitor-treated embryos. Red spots represent BrdU signal. HCs are labeled with an antibody against myosin VI (green). Scale bar=10μm. (b) Quantification of replicating cells in the neuromasts for each experimental condition. The BrdU index is higher in the control relative to the HDAC inhibitor-treated fish. The proliferating cells of the first four neuromasts along the body, L1 to L4, were recorded on one side of each fish. Bars are meanąs.e.m. and n=36 neuromasts per condition. **P<0.001. (c) Confocal images of neuromasts from a 5dpf control and from 5dpf embryos treated with 0.1μM TSA, 100μM VPA or 10μM MS-275. HDAC inhibitor treatment decreased the numbers of both BrdU-positive HCs and BrdU-positive SCs in neuromasts. White arrows indicate HCs that were derived from proliferating SCs. Scale bar=10μm. (d, e) Quantification of replicating cells co-labeled with Myosin VI or Sox2 per neuromast (NM) in control and HDAC inhibitor-treated larvae. Bars are meanąs.e.m. and n=36 neuromasts per condition. **P<0.001. (f) Real-time RT?PCR analysis showed that TSA treatment increases the mRNA expression of p21, p27 and p53. The bars represent the meansąs.d. of the ratios of p21, p27 and p53 to GAPDH in the real-time RT?PCR data. *P<0.05.