Fig. 1

GSK-J4 impaired hair cell regeneration after neomycin damage. (A–D) GSK-J4 reduced the numbers of GFP-positive (green) and FM1-43FX-positive (red) hair cells compared with DMSO-treated controls. Scale bars = 10 μm. (E,F) Quantitative analysis of the number of GFP-positive (E) or FM1-43FX-positive (F) hair cells per neuromast (NM) at different time points in DMSO-treated control and GSK-J4-treated larvae. In the 24-h group, n = 40 neuromasts (20 larvae) per group; in the 48-h group, n = 28 neuromasts (14 larvae) per group. ***p < 0.0001. Bars are mean ± sem. [24-h group: One-way ANOVA; GFP-positive cells: F(2, 117) = 96.94; FM1-43FX-positive cells: F(2, 117) = 114. 48-h group: One-way ANOVA; GFP-positive cells: F(2, 81) = 88.96; FM1-43FX-positive cells: F(2, 81) = 93.85].
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.
Fig. 3

GSK-J4 induced apoptosis in the regenerating neuromasts. (A,B) Cleaved caspase-3 staining in the neuromasts from a DMSO-treated control larva (A) and a GSK-J4-treated larva (B) at 48 h after neomycin damage. Scale bar = 10 μm. (C) Quantitative analysis of the number of cleaved caspase-3-labeled cells in DMSO-treated control and GSK-J4-treated larvae. n = 24 neuromasts (12 larvae) per group. **p < 0.001 (unpaired t-test, two-tailed, t = 3.368, df = 44, p = 0.0016). (D) Cleaved-caspase-3-specific western blot analysis of whole larvae treated with either DMSO (neo Con; n = 6 larvae) or 10 μM GSK-J4 (neo GSK-J4; n = 6 larvae) for 48 h. The relative expressions of cleaved-caspase-3/β-actin were calculated. **p < 0.001.
Fig. 4

GSK-J4 increased the levels of H3K27me3. (A,B) Immunohistochemistry results showing H3K27me3 expression in neuromasts of DMSO-treated control (Con) larvae (n = 5 larvae) and of 10 μM GSK-J4-treated larvae (n = 6 larvae) for 48 h. Scale bar = 10 μm. (C) Western blot analysis was performed to evaluate the expressions of H3K27me3 and β-actin in whole larvae that were treated with either DMSO (neo Con; n = 6 larvae) or GSK-J4 (neo GSK-J4; n = 6 larvae) for 48 h. *p < 0.05.
Fig. S1

Five dpf zebrafish larvae treated with 400 μM neomycin to kill mature lateral line hair cells.
Fig. S2

GSK-J1 impaired zebrafish hair cell regeneration. (A–F) 5 dpf larvae were treated with 400 μM neomycin for 1 h followed by GSK-J1 exposure for 24 or 48 h in the presence of BrdU. GSK-J1 significantly reduced the numbers of myosinVI-positive (green) hair cells and BrdU-positive (red) replicating cells. Scale bars = 10 μm. (G,H) Quantification of myosinVI-positive and BrdU-positive cells per neuromast (NM) in DMSO-treated control larvae (Con), 15 μM GSK-J2-treated control larvae, and 15 μM GSK-J1-treated larvae at 24 or 48 h following neomycin damage. In the 24-h group, n = 28 neuromasts of DMSO-treated control larvae (14 larvae), n = 30 neuromasts of 15 μM GSK-J2-treated control larvae (15 larvae), and n = 20 neuromasts of 15 μM GSK-J1-treated larvae (10 larvae); in the 48-h group, n = 18 neuromasts of DMSO-treated control larvae (9 larvae), n = 22 neuromasts of GSK-J2-treated control larvae (11 larvae), and n = 18 neuromasts of 15 μM GSK-J1-treated larvae (9 larvae). ***p < 0.0001. Bars are mean ± sem.
Fig. S3

GSK-J4 incubation did not affect the pattern of cell proliferation during development. (A,B) In the larvae not exposed to neomycin, supporting cell proliferation was at a low level overall for both DMSO-treated control and GSK-J4-treated larvae. (C,D) Quantification of myosinVI-positive hair cells and BrdU-positive cells per neuromast (NM) in DMSO-treated 5 dpf control larvae (Con) and 10 μM GSK-J4-treated 5 dpf larvae for 24 h. n = 20 neuromasts of DMSO vehicle control larvae (10 larvae) and n = 28 neuromasts of 10 μM GSK-J4-treated larvae (14 larvae).
Fig. S4

Effects of GSK-J4 on apoptosis in the zebrafish body. Detection of cell apoptosis by cleaved caspase-3 staining in the body of zebrafish larvae exposed to DMSO (Con) or 10 μM GSK-J4 at 48 h following neomycin damage. The cleaved caspase-3-positive cells are indicated by white arrows, and the neuromasts are outlined.
Fig. S5

Effects of ERK1/2 inhibition on the expression of p21 and p27. The mRNA levels of p21 and p27 in regenerating neuromasts were increased after U0126 treatment at 12 hpt when compared to the respective control larvae (n = 16–20 neuromasts per group).
Acknowledgments
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