Fig 1: Western blot analysis of mitochondrial proteins. (A) Twenty μg of total proteins from mutant and wild type zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies for 6 subunits of OXPHOS (four encoded by mtDNA and two encoded by nuclear genes), Kars, Lars2, Yars2, Eftu, Tfb2m as well as Tom20 as a loading control. Quantification of levels of OXPHOS subunits (B) and other mitochondrial proteins (C). Average content of Nd1, Nd6, Cytb, Co2 and Atp5a, Sdhb, Kars, Lars2, Yars2, Eftu and Tfb2m was normalized to the average content of Tom20 in mutant and wild type zebrafish. The values for the mutant zebrafish are expressed as percentages of the average values for the wild type zebrafish. The horizontal dashed lines represent the average values of four proteins encoded by mtDNA or two proteins encoded by nuclear genes for each group. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to the Figure 3.
Fig 2: Mitochondrial defects in hair cells. (A) Assessment of mitochondrial function in hair cells by enzyme histochemistry (EHC) staining for SDH and COX in the frozen-sections of posterior macula of mtu1−/−, mtu1+/− and mtu1+/+ zebrafish at 5dpf. Loss of EHC signal is indicated by arrows (magnification X400). V, ventral; L, lateral; Ov, otic vesicle; Hc, hair cell; Sc, supporting cell. (B) Mitochondrial networks from hair cells of inner electron microscopy. Ultrathin sections were visualized with 5000×, 10 000×, 20 000× and 60 000× magnifications. (C) Quantification of mitochondrial numbers of hair cells from the mtu1−/−, mtu1+/− mutant and mtu1+/+ zebrafish. The calculations were based on 50 different hair cells of mtu1−/−, mtu1+/− mutant and mtu1+/+ zebrafish, respectively. Graph details and symbols are explained in the legend to Figure 3.
Fig 3: Hypertrophic cardiomyopathy and mitochondrial defects in the zebrafish. (A) Hematoxylin and eosin stained (H&E) histological sections of hearts from mto1−/−, mto1+/− and mto1+/+ zebrafish at the age of 6 months. Scale bars: 20 μm. (B) The relative cross-sectional areas of cardiomyocytes in the mto1+/+ (n = 104), mto1+/− (n = 112), mto1−/− (n = 92) zebrafishes, staining with H&E. (C) The cardiomyocytes from mutant and WT zebrafishes were visualized via WGA staining. Scale bars: 20 μm. (D) Ventricular heart muscle sections of transmission electron microscopy in the mutant and WT zebrafish. Ultrathin sections were visualized with 15 000× magnifications. Widened I-band in the sarcomere units were indicated by white arrowhead. Scale bars: 1 μm. (E) Quantification of the I-band lengthes of mto1+/+ (n = 28), mto1+/− (n = 18) and mto1−/− (n = 29) zebrafish. The values for the mutants were expressed as percentages of the mean values for the WT. (F) Assessment of mitochondrial functions in cardiomyocytes by enzyme histochemistry (EHC) staining for cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) in the frozen-sections of ventricles in the mto1−/−, mto1+/− and mto1+/+ zebrafish at six-month old. Scale bars: 50 μm. (G) Mitochondrial networks from cardiomyocytes of transmission electron microscopy. Ultrathin sections were visualized with 30 000× magnifications. Fragmented mitochondria are indicated by asterisks. Scale bars: 0.5 μm. (H) Quantifications of fragmented mitochondria numbers of cardiomyocytes from the mto1−/−, mto1+/− and mto1+/+ zebrafish. The error bars indicate standard deviations of the means. P indicates the significance, according to Student's t test, of the difference between mto1+/− or mto1−/− and WT values, denoted by asterisks (*P< 0.05, **P< 0.01, ***P< 0.001), and non-significant differences by n.s.
Supplier Page from Abcam for Anti-SDHB antibody