Figure 6.

CoQ₁₀ administration rescued nephrocyte functional and ultrastructural defects induced by Coq2 gene silencing. (A) Fluorescence micrographs showing nephrocytes of 1-day postemergence adult flies. Flies were reared from embryos on standard food supplemented with the indicated concentrations (1% or 5%) of CoQ₁₀ (Q₁₀). Left panels (MHC-ANF-RFP) show intracellular ANF-RFP fluorescence (red). Right panels (MHC-ANF-RFP Hand-GFP) show RFP (red) merged with GFP (green, mostly nuclear). A GFP transgene is expressed under the control of a Hand gene enhancer (Hand-GFP) to confirm pericardial nephrocyte cell identity.²³ All flies are transgenic for Hand-GFP. Control flies carry the Dot-Gal4 driver but no RNAi construct. Coq2 flies carry Dot-Gal4 driving RNAi transgene silencing Coq2 expression. (B) Quantitation of RFP levels in control versus Coq2-IR nephrocytes (expressed relative to control) with no Q₁₀, 1% Q₁₀, or 5% Q₁₀ dietary supplementation. For each genotype, 30 nephrocytes (six nephrocytes from each of five flies) were examined (*P<0.05). (C) TEM showing mislocalized slit diaphragms and irregularly spaced and collapsed lacunar channel ultrastructure induced by Coq2 silencing (upper panel) rescued by administration of 5% Q₁₀ (lower panel). Scale bar, 300 nm. (D) Quantitation of ectopic slit diaphragms in Coq2-IR versus Coq2-IR plus 5% Q₁₀ supplementation nephrocytes. Average number of slit diaphragms positioned along interior channel membranes per 2000 nm length of cell circumference (*P<0.05). (E) The average distance (in nm) between normally localized slit diaphragms in Coq2-IR versus Coq2-IR plus 5% Q₁₀ supplementation nephrocytes (*P<0.05). (F) ROS levels in normal (control) and Coq2-silenced (Coq2) nephrocytes were indicated by oxidized DHE red fluorescence in the cell nucleus. ROS levels were higher in Coq2-IR nephrocytes, and feeding Coq2-IR larvae a diet supplemented with 5% Q₁₀ reduced nephrocyte ROS levels. Dashed lines indicate cell nuclei (determined from DAPI staining, not shown). (G) Levels of DHE red nuclear fluorescence expressed relative to normal control larval nephrocytes fed a nonsupplemented diet. Coq2 gene silencing led to a 2.5–3-fold increase in ROS levels. Feeding Coq2-IR 5% Q₁₀ lowered ROS to normal levels. In each case, 30 nephrocytes (six nephrocytes from each of five larvae) were examined (*P<0.05).