To knock down Twist TFs, lissamine-tagged MOs (red) against twist 1a, 1b, 2, and 3 were microinjected into Tg(α-actin::EGFP) (green) fish muscles 4 h prior to myectomy. (A-E'') MO injected fish were shown. (H-J) Knockdown effect of twist 1a/b, and twist2 MO was validated in embryos by Western blot. (K) Knockdown effect of twist3 MO was validated in EOM by Western blot. (F) The length of regenerating muscle was measured as described; values are averages ±SEM (one-way ANOVA, p<0.05 between group a and b, control:n = 6, twist1a: n = 4, twist1b: n = 5, twist2: n = 6, twist3: n = 7). (G) Twist3 MO inhibits but does not delay EOM regeneration; mixture of all 4 Twist TFs MOs or single control and twist3 MO were microinjected into α-actin-EGFP fish muscles 4 h prior to myectomy. The length of regenerating muscle was measured as described; values are averages ±SEM (one-way ANOVA, p<0.05 between group a and b, control: n = 8, twist1/2/3: n = 4, twist3: n = 5). White arrows marked the growing end of the regenerating muscle. p, pituitary; e, eye; scale bar: 250μm.
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