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Fig. 2
GSK-J4 reduced proliferation in regenerating neuromast cells. (A–D) 5 dpf larvae were treated with 400 μM neomycin for 1 h followed by GSK-J4 exposure for 24 or 48 h in the presence of BrdU. GSK-J4 significantly reduced the numbers of Sox2-positive (red) and BrdU-positive (white) replicating cells. Scale bars = 10 μm. (E,F) Quantification of Sox2-positive and BrdU-positive cells per neuromast (NM) in DMSO-treated control larvae and 10 μM GSK-J4-treated larvae at 24 or 48 h following neomycin damage. In the 24-h group, n = 30 neuromasts of DMSO-treated control larvae (15 larvae) and n = 24 neuromasts of 10 μM GSK-J4-treated larvae (12 larvae); in the 48-h group, n = 36 neuromastsof DMSO-treated control larvae (18 larvae) and n = 24 neuromasts of 10 μM GSK-J4-treated larvae (12 larvae). ***p < 0.0001. (24-h group: Sox2-positive cells: unpaired t-test, two-tailed, t = 7.412, df = 52; BrdU-positive cells: unpaired t-test, two-tailed, t = 13.86, df = 52. 48-h group: Sox2-positive cells: unpaired t-test, two-tailed, t = 7.463, df = 58; BrdU-positive cells: unpaired t-test, two-tailed, t = 15.6, df = 58). Bars are mean ± sem. (G,H) Quantitative analysis of the proportion of BrdU-positive hair cells (G) or BrdU-positive supporting cells (H) in control and GSK-J4-treated larvae at 24 or 48 h after neomycin damage. In the 24-h group, n = 30 neuromasts of DMSO-treated control larvae (15 larvae) and n = 24 neuromasts of 10 μM GSK-J4-treated larvae (12 larvae); in the 48-h group, n = 36 neuromasts of DMSO-treated control larvae (18 larvae) and n = 24 neuromasts of 10 μM GSK-J4-treated larvae (12 larvae). ***p < 0.0001. (24-h group: BrdU-positive HCs: unpaired t-test, two-tailed, t = 5.309, df = 52; BrdU-positive SCs: unpaired t-test, two-tailed, t = 6.294, df = 52. 48-h group: BrdU-positive HCs: unpaired t-test, two-tailed, t = 7.279, df = 58; BrdU-positive SCs: unpaired t-test, two-tailed, t = 9.546, df = 58). Bars are mean ± sem. (I) Localization of the p21 and p27 genes by whole-mount in situ hybridization in GSK-J4-treated and DMSO-treated control larvae. GSK-J4 treatment significantly increased the expression of p21 and p27 in regenerating neuromasts at 12 hpt. (n = 20–26 neuromasts per group). Results from single representative neuromasts are shown.