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Fig. 5
Exogenous HSP60 promotes hair cell regeneration and signalling inhibitor L-37pA inhibits regeneration. (a) GroEL injected into the trunk attracted mpx-positive neutrophils similar to brain ventricle injections. The areas framed with red dotted lines in the left panels indicate the areas of injection (the panel on the left) or quantification (three panels on the right). The red colour shown within the dotted lines of the first panel is from phenol red in the injection buffer. BSA protein is used as a control for GroEL protein and also a control for assessing the needle-induced injury. Red arrows point to the sites of injection. (b) GroEL injected into the trunk promotes hair cell regeneration. The top panel shows a 7 dpf zebrafish larvae stained with Yopro-1, with each fluorescent dot indicating a hair cell containing neuromast. The injection site is pointed. The bottom panels show representative images of hair cells in a neuromast for each condition. (c) Quantification of hair cell sensitivity to copper (left) and rate of regeneration (right). GroEL does not provide protection from cell death (n=16, P=0.471), but does significantly boost regeneration rates (n=15, P<0.001). (d) Quantification of hair cell regeneration stimulated by human HSP60. Human HSP60 injected into the trunk promotes hair cell regeneration significantly (n=34 for GAPDH, n=36 for human HSP60, P<0.001). (e) L-37pA injected into the trunk before hair cell ablation inhibits hair cell regeneration (n=8, P=0.002). Injection of 50 or 100 pg of L-37pA inhibits regeneration to a similar degree (n=8, P=0.176). (f) L-37pA injected into the trunk 1 day after hair cell ablation inhibits hair cell regeneration (n=13, P=0.01). For all experiments, hair cells were ablated in WT embryos at 5 dpf, and then hair cell regeneration evaluated at 7 dpf. (g) LPS injected into the trunk does not promote hair cell regeneration. Injection of 75 pg of LPS does not affect hair cell regeneration (n=8, P=0.938), whereas injection of 150 pg of LPS caused a slight but significant inhibition of regeneration (n=8, P=0.023). Asterisks in the graphs indicate a significant difference between the indicated two groups. Bars = 200 μm in (a), 10 μm in (b). dpf, days post-fertilisation. BSA, bovine serum albumin; HCR, hair cell regeneration; HCS, hair cell sensitivity; LPS, lipidpolysaccharide; WT, wild type.