Figure 4

Representative electron micrographs from (A) wild-type/heterozygous sibling control and (B) vma21^Δ1/Δ1 zebrafish larvae skeletal muscle at 6 days post fertilization (dpf), showing the presence of double-membrane vacuoles in vma21 mutants but not controls. Scale bars: 2 mm. (C) Representative western blot of LC3I, LC3II, and b-actin (housekeeping protein) protein levels in the vma21 1 bp deletion (vma21Δ1/Δ1) and the 14 bp deletion with 21 bp insertion (vma21Δ14ins21/Δ14ins21) homozygous mutants as well as the compound heterozygous line (vma21Δ1/Δ 14ins21) compared to their respective wild-type/heterozygous sibling controls at 5 days post fertilization. LC3I protein is ~19 kDa, LC3II protein is approximately 17 kDa, and b-actin is approximately 42 kDa. Each lane represents n = 10 zebrafish (50 mg of total protein). Each western blot had a total of three biological replicates. (D) LC3I and LC3II protein levels were quantified by normalization to b-actin loading controls. The LC3II/LC3I ratio was also determined. For each sample, a total of n = 30 zebrafish were utilized. Data shown as mean ± SEM. Significance at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA). Representative fluorescent images of (E, F) wild-type/heterozygous sibling control and (G, H) vma21^Δ1/Δ1 zebrafish larvae in GFP and RFP channels at 5 dpf following injection of 1-cell stage embryos with the pTol2 (Ubbi: GFP-LC3-RFP-LC3ΔG) construct used to assess autophagic flux. Scale bar: 500 mm. (I) GFP/RFP ratio as a measure of autophagic flux in 5-dpf sibling control and vma21^Δ1/Δ1. n = 15–17/group. Data represented as mean ± SEM. Significance at ***P < 0.001 (unpaired two-tailed t test). Source data are available online for this figure.