Zebrafish injected with fut8a MO showed abnormalities in skeletal muscle (a) Lectin blot, with AAL, of Zebrafish morphants injected with 3 ng of CMO or fut8a MO at 2 dpf. Total protein was visualized by Coomassie Blue staining and Ponceau staining (Supplemental Figure S1a,b): Histogram of the percentage of normal, abnormal, and dead fish in non-injected fish, CMO and fut8a MO injected fish at 2 dpf. Blue: normal, orange: abnormal, gray: dead. (c,d) Comparison of the CMO and fut8a MO injected zebrafish under bright field (c) and birefringence assay (d) at 2 dpf. d: Upper bar = 1 mm; lower bar = 200 μm. Non: non-injected zebrafish.

Reduced fut8a in zebrafish triggers skeletal abnormalities anti-laminin, anti-beta-dystroglycan (βDG), anti-myosin heavy chain (Myosin HC), and DAPI. Scale bar: 50 μm.

fut8a reduction causes structural abnormalities in myosepta (a) Left panel is CMO, right panel is fut8a MO. The white dotted line indicates the angle between the dorsal vertical and transverse myoseptas, and the double arrow indicates the distance of the transverse myoseptas, stained with anti-laminin. Scale bar: 50 μm. (b) Histogram of the angle between the vertical and transverse myoseptas. (c) Histogram of the variation in sarcomere length. Five to ten sarcomere lengths were calculated from five fish. CMO: Median 1.956, Max 2.032, Min 1.869. fut8a MO: Median 2.0325, Max 2.153, Min 1.772 (t-test. p < 0.01, n = 5).

fut8a morphants have abnormal myosepta Representative electron micrographs of longitudinal section of morphants (2 dpf). (a) Electron micrograph of dorsal myosepta. White arrows indicate the tear sites in the myosepta. Scale bar: 5 μm. (b) Enlarged views of electron micrograph of dorsal myosepta. Scale bar: 1 μm. Maximum width, total length, and straightness factor of myosepta were quantified and indicated in Supplemental Figure S2.

fut8a morphants show abnormalities in sarcomere formation (a) fut8a morphants displayed abnormal sarcomere structure with distorted Z-line and H-bands. Scale bar: 1 μm. (b) The triad of fut8a morphants was disorganized. Black boxes show the location of lower images. Scale bar: 1 μm. (c) Histogram of the variation in sarcomere length. CMO: Median 1.956, Max 2.032, Min 1.869. fut8a MO: Median 2.0325, Max 2.153, Min 1.772 (t-test. p < 0.01, n = 5).

Altered expression and fucosylation of Fut8 in C2C12 cells (a) Fut8a expression during C2C12 cell differentiation. Relative control normalized by r18 on day 1. (b) Lectin blot, with AAL and anti-β-actin, of C2C12 cells with induced muscle differentiation. (c) Lectin blot, with AAL and anti-α-tubulin, of C2C12 cells treated with 1 mM 2-Fluorofucose (2FF). * Nonspecific band.

Inhibition of Fut8 function by 2FF inhibits myotube formation (a) Changes in muscle differentiation after the addition of 0, 0.5, and 1 mM 2FF. Scale bar: 100 μm (b) Immunostaining of C2C12 cells on day 5 of induced muscle differentiation using anti-MY32. Scale bar: 100 μm. (c) Diameter of myotube (one-way ANOVA, p < 0.01, n = 3). (d) Length of myotube (one-way ANOVA, p < 0.01, n = 3). (e) Histogram of fusion index (one-way ANOVA, p < 0.05). (f) Differentiation index (one-way ANOVA, p < 0.05). (g–j) Quantitative RT-PCR using the delta-delta Ct method for Myomaker, Myomerger, Ckm, and Myogenin expression during differentiation of C2C12 cells. Relative control normalized by r18 and Day 0 (n = 3).