Ccn6 depletion inhibits mitochondrial respiratory complex assembly and activity in zebrafish muscle. (A,B) Immunoblotting of the total muscle lysate and corresponding band densitometry demonstrating about 60% reduction in Ccn6 expression by ccn6 morpholino, but not control morpholino, as compared to the corresponding uninjected control. β-actin is used as a reference for the estimation of Ccn6 expression. (C–F) Blue native (BN)-PAGE of the muscle mitochondrial lysate followed by (i) immunoblotting separately with antibodies to Ndufb8 (complex I), Uqcrc2 (complex III), Cox4 (complex IV), and Atp5a1 (complex V) and (ii) corresponding band densitometry projected by a distribution plot, demonstrating that ccn6 morpholino, but not non-targeted morpholino, inhibits respiratory complex assembly as compared to no injection. Vdac1 expression from equal amounts of mitochondrial protein is used as a reference for this estimation. (G,H) Spectrophotometric and bar graph representation of the decrease in complex I activity upon Ccn6 depletion by ccn6 morpholino, but not control morpholino. No injection is a reference for this estimation. (I) ATP measurement assay demonstrating the decrease in ATP synthesis upon ccn6 morpholino (MO), but not control MO injection, as compared to the uninjected control. Data are presented as the mean ± SEM of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not significant (Student’s t-test). For pooled experiments (C–I), the number of fish used were 10, 10, 10, and 5, respectively, in each experimental group.
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