Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q 10 Supplementation

doi: 10.1681/ASN.2016060626

Figure Lengend Snippet: Genes involved in CoQ biosynthesis and associated with clinical renal pathology

Article Snippet: The wild-type COQ2 cDNA was obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: {"type":"entrez-protein","attrs":{"text":"AAH08804","term_id":"14250676","term_text":"AAH08804"}} AAH08804 ).

Techniques: Modification

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q 10 Supplementation

doi: 10.1681/ASN.2016060626

Figure Lengend Snippet: Hemolymph protein marker ANF-RFP and AgNO3 levels in nephrocytes expressing Coq-RNAi transgenes. (A) Fluorescence micrographs showing nephrocytes of adult flies 1-day postemergence. ANF-RFP fluorescence (red) is shown in the left panels. Right panels 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. Coq-IR flies carry Dot-Gal4 driving an RNAi transgene to silence expression of Coq2, Coq6, or Coq8 genes. (B) Quantification of nephrocyte RFP fluorescence, expressed relative to control value. For each genotype, 30 nephrocytes (six nephrocytes from each of five flies) were examined (*P<0.05). (C) Photomicrographs showing nephrocytes of third instar larvae reared on standard fly food supplemented with AgNO3. Control flies carry the Dot-Gal4 driver but no RNAi construct. Coq-IR flies carry Dot-Gal4 driving an RNAi transgene to silence expression of the indicated Coq gene. Scale bar, 20 microns. (D) Quantification of AgNO3, expressed relative to control value. For each genotype, 30 nephrocytes (six nephrocytes from each of five larvae) were examined (*P<0.05).

Article Snippet: The wild-type COQ2 cDNA was obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: {"type":"entrez-protein","attrs":{"text":"AAH08804","term_id":"14250676","term_text":"AAH08804"}} AAH08804 ).

Techniques: Marker, Expressing, Fluorescence, Control, Transgenic Assay, Construct

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q 10 Supplementation

doi: 10.1681/ASN.2016060626

Figure Lengend Snippet: Coq2 gene silencing induced abnormal slit diaphragm localization and collapsed lacunar channels with multiple slit diaphragms (A) TEM showing normal (control) nephrocyte ultrastructure with slit diaphragms and lacunar channels uniformly spaced along the circumference of the cell. Slit diaphragms localized exclusively at the mouth of the channel. In Coq2-IR nephrocytes, channels appeared narrower (collapsed) and interchannel spacing was interrupted and irregular. Slit diaphragms occurred not only at the channel mouth but also ectopically along the interior channel membranes. Scale bar, 200 nm. (B) Higher magnification TEM comparing normal control and Coq2-IR slit diaphragm and lacunar channel ultrastructure. Ectopic slit diaphragms arranged in ladder-like configuration at points of channel narrowing are indicated by arrows. Scale bar, 100 nm. (C) Quantitation of normally localized slit diaphragms in control versus Coq2-IR nephrocytes. Average number of slit diaphragms positioned at mouths of lacunar channels per 2000 nm of cell circumference (*P<0.05). (D) The average distance (in nm) between normally localized slit diaphragms in control versus Coq2-IR nephrocytes (*P<0.05). (E) Quantitation of ectopic slit diaphragms in control versus Coq2-IR nephrocytes. Average number of slit diaphragms positioned along interior channel membranes per 2000 nm of cell circumference (*P<0.05). (F) Quantitation of slit diaphragms (normally localized plus ectopic) in control versus Coq2-IR nephrocytes. Total number of slit diaphragms per 2000 nm of cell circumference (*P<0.05).

Article Snippet: The wild-type COQ2 cDNA was obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: {"type":"entrez-protein","attrs":{"text":"AAH08804","term_id":"14250676","term_text":"AAH08804"}} AAH08804 ).

Techniques: Control, Quantitation Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q 10 Supplementation

doi: 10.1681/ASN.2016060626

Figure Lengend Snippet: Coq2 gene silencing induced elevated numbers of autophagosomes and abnormal mitochondria in nephrocytes. (A) Upper panels: TEM showing normal mitochondria (m) in control nephrocyte of a wild-type fly. In Coq2-IR nephrocytes the mitochondria are more abundant and morphologically abnormal. Autophagosomes (ap) are in evidence. Scale bar, 300 nm. Lower panels: higher magnification electron micrographs comparing normal control and Coq2-IR nephrocyte mitochondria. In nephrocytes in which Coq2 expression was silenced the mitochondria exhibited shortened cristae and fewer inner mitochondrial membranes. Scale bar, 200 nm. (B) Quantitation of mitochondria in control versus Coq2-IR nephrocytes. Average number of mitochondria per 4 μm2 area of cytoplasm (*P<0.05). (C) Percentage of mitochondria exhibiting abnormal morphology in control versus Coq2-IR nephrocytes (*P<0.05). (D) TEM showing mitochondria and autophagosomes in control nephrocyte of a wild-type fly. In Coq-IR nephrocytes, the number of autophagosomes was increased. Scale bar, 300 nm. (E) Quantitation of autophagosomes in control versus Coq2-IR nephrocytes. Average number of autophagosome per 4 μm2 area of cytoplasm (*P<0.05).

Article Snippet: The wild-type COQ2 cDNA was obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: {"type":"entrez-protein","attrs":{"text":"AAH08804","term_id":"14250676","term_text":"AAH08804"}} AAH08804 ).

Techniques: Control, Expressing, Quantitation Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q 10 Supplementation

doi: 10.1681/ASN.2016060626

Figure Lengend Snippet: Coq2 gene silencing induced autophagy and mitophagy. (A) Fluorescence micrographs showing nephrocytes of adult female flies 1-day postemergence, expressing a dual-labeled GFP-Atg8-mCherry autophagy marker protein specifically in nephrocytes (UAS-GFP-Atg8-mCherry construct driven by Dot-Gal4). Dotted lines indicate nephrocyte cell boundary. Atg8 is present in autophagosomes and subsequently accumulates in autolysosomes. Fluorescence from the compartmentalized marker protein is punctate in appearance. Both GFP and mCherry fluorescence can be detected in autophagosomes. In the acidic lysosome lumen, GFP fluorescence undergoes rapid quenching whereas mCherry retains fluorescence activity. Therefore only very early autolysosomes will transiently exhibit GFP fluorescence. Because Atg8 accumulates to high levels in autolysosomes, mCherry fluorescence is relatively stronger in this compartment. Upper panels show control nephrocytes (normal Coq2 gene expression) in which autophagy is essentially undetectable. Lower panels show nephrocytes in which Coq2 gene expression was silenced (Coq2-IR) in which a few autophagosomes were present (GFP, green; GFP-Atg8-mCherry, yellow) and autolysosomes were highly abundant (mCherry, red). (B) Fluorescence micrographs showing nephrocytes of adult female flies 1-day postemergence, expressing GFP-labeled mitochondria and Atg8-RFP marker protein in nephrocytes. Dotted line indicates nephrocyte cell boundary. Upper panels show control nephrocytes (normal Coq2 gene expression) with labeled mitochondria (Mito-GFP, green), essentially undetectable autophagy (Atg8-RFP, red), and no overlapping yellow fluorescence in merged images (Mito-GFP Atg8-RFP). Lower panels show nephrocytes in which Coq2 was silenced (Coq2-IR) with increased mitochondrial fluorescence (higher Mito-GFP fluorescence, green), fluorescence because of Atg8 in autophagosomes/autolysosomes (Atg8-RFP, red), and overlap of fluorescence indicating mitophagy (Mito-GFP Atg8-RFP, yellow). (C) Quantitative comparison of Mito-GFP fluorescence in control versus Coq2-IR nephrocytes, expressed relative to control. The increased fluorescence due to Coq2 silencing is shown relative to control. For each group, 30 nephrocytes (six nephrocytes from each of five flies) were examined (*P<0.05). (D) Quantitative comparison of Atg8-RFP fluorescence in control versus Coq2-IR nephrocytes, expressed relative to Control. For each group, 30 nephrocytes (six nephrocytes from each of five flies) were examined (*P<0.05). (E) Quantitative spatial comparison of Mito-GFP (green), Atg8-RFP (red), and Mito-GFP Atg8-RFP (yellow) fluorescence in control versus Coq2-IR nephrocytes.

Article Snippet: The wild-type COQ2 cDNA was obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: {"type":"entrez-protein","attrs":{"text":"AAH08804","term_id":"14250676","term_text":"AAH08804"}} AAH08804 ).

Techniques: Fluorescence, Expressing, Labeling, Marker, Construct, Activity Assay, Control, Gene Expression, Comparison

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q 10 Supplementation

doi: 10.1681/ASN.2016060626

Figure Lengend Snippet: Simultaneous silencing of Coq2 and autophagy genes induces synergistic effects. (A) Fluorescence micrographs showing nephrocytes of 1-day postemergence adult flies transgenic for Hand-GFP (GFP expressed under the control of a Hand gene enhancer to confirm pericardial nephrocyte cell identity).23 ANF-RFP (red) is merged with GFP (green, predominantly in nucleus). Control flies carry the Dot-Gal4 driver but no RNAi construct. Gene-IR flies carry Dot-Gal4 driving RNAi transgenes silencing endogenous Coq2, Atg1, or Atg6 genes singly or in combination. (B) Quantitation of nephrocyte RFP levels, relative to control. For each genotype, 30 nephrocytes (six nephrocytes from each of five flies) were examined (* indicates significance compared with control, # indicates significance compared with Coq2-IR; P<0.05). (C) Oxidized DHE fluorescence (red) in nephrocytes of third instar larvae. Dashed lines indicate cell nuclei (determined from DAPI staining, not shown). (D) Quantitation of DHE fluorescence intensity expressed relative to control nephrocytes. For each genotype, 30 nephrocytes (six nephrocytes from each of five larvae) were examined (* indicates significance compared with control, # indicates significance compared with Coq2-IR; P<0.05).

Article Snippet: The wild-type COQ2 cDNA was obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: {"type":"entrez-protein","attrs":{"text":"AAH08804","term_id":"14250676","term_text":"AAH08804"}} AAH08804 ).

Techniques: Fluorescence, Transgenic Assay, Control, Construct, Quantitation Assay, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q 10 Supplementation

doi: 10.1681/ASN.2016060626

Figure Lengend Snippet: 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).

Article Snippet: The wild-type COQ2 cDNA was obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: {"type":"entrez-protein","attrs":{"text":"AAH08804","term_id":"14250676","term_text":"AAH08804"}} AAH08804 ).

Techniques: Functional Assay, Fluorescence, Control, Transgenic Assay, Construct, Expressing, Quantitation Assay, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q 10 Supplementation

doi: 10.1681/ASN.2016060626

Figure Lengend Snippet: A normal allele of the human COQ2 gene but not a mutant allele (COQ2-S146A) rescued nephrocyte function in flies expressing Drosophila Coq2-IR. (A) Fluorescence micrographs showing uptake of ANF-RFP nephrocytes of 1-day postemergence adult flies. 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). Hand-GFP expression confirms pericardial nephrocyte cell identity. All flies are transgenic for Hand-GFP. Control flies carry the Dot-Gal4 driver but no RNAi construct. Coq2-IR flies carry Dot-Gal4 driving RNAi transgene silencing the endogenous Drosophila Coq2 gene expression, and where indicated, also a UAS-COQ2 (wild-type human COQ2 allele) or a UAS-COQ2-S146N (mutant human COQ2 allele).6 (B) Quantitation of RFP levels (expressed relative to control) in control versus Coq2-IR nephrocytes expressing normal COQ2 or mutant COQ2-S146N. For each genotype, 30 nephrocytes (six nephrocytes from each of five flies) were examined (*P<0.05).

Article Snippet: The wild-type COQ2 cDNA was obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: {"type":"entrez-protein","attrs":{"text":"AAH08804","term_id":"14250676","term_text":"AAH08804"}} AAH08804 ).

Techniques: Mutagenesis, Expressing, Fluorescence, Transgenic Assay, Control, Construct, Gene Expression, Quantitation Assay

Journal: Molecular Medicine Reports

Article Title: Collagen III regulates the termination of liver regeneration by suppressing hepatocyte proliferation and promoting functional recovery

doi: 10.3892/mmr.2026.13799

Figure Lengend Snippet: Liver regeneration status after collagen III degradation during the termination phase of liver regeneration. (A) Body weight of mice. (B) Liver-to-body weight ratio of mice. (C) Dual IF staining of EdU and Ki-67. (D) Dual IF staining of EdU and collagen III. Scale bars, 100 and 10 µm. *P<0.05, **P<0.01 and ***P<0.001. ns, not significant; PHx, partial hepatectomy; IF, immunofluorescence; Coln0.1, low-dose collagenase III; Coln0.2, high-dose collagenase III; Col III, collagen III.

Article Snippet: For the high-dose collagenase + MSAB group (Coln0.2 + MSAB group), 5 mg/kg/day MSAB (cat. no. HY-120697; MedChemExpress) was dissolved in an ultrasonic ice bath and administered intraperitoneally 2 h prior to each collagenase injection.

Techniques: Staining, Immunofluorescence

Journal: Molecular Medicine Reports

Article Title: Collagen III regulates the termination of liver regeneration by suppressing hepatocyte proliferation and promoting functional recovery

doi: 10.3892/mmr.2026.13799

Figure Lengend Snippet: Inhibition of β-catenin partially rescues impaired regeneration termination and liver dysfunction induced by collagen III degradation. (A) Schematic diagram of the animal experimental protocol; created with BioGDP.com (agreement no. GDP2025X2AT2F) . (B) WB analysis of β-catenin after collagen III degradation. (C) Body weight and (D) liver-to-body weight ratio of mice following collagen III degradation and β-catenin inhibition. (E) Dual immunofluorescence staining of EdU and collagen III following collagen III degradation and β-catenin inhibition. Scale bars, 100 and 10 µm. (F) Serum liver function marker levels following collagen III degradation and β-catenin inhibition. (G) WB analysis of HNF4α following collagen III degradation and β-catenin inhibition. (H) WB analysis of cyclin D1 following collagen III degradation and β-catenin inhibition. *P<0.05, **P<0.01 and ***P<0.001. ns, not significant; WB, western blot; PHx, partial hepatectomy; MSAB, methyl-sulfonyl AB; Coln0.1, low-dose collagenase III; Coln0.2, high-dose collagenase III; Col III, collagen III; ALT, alanine aminotransferase; HNF4α, hepatocyte nuclear factor 4α.

Article Snippet: For the high-dose collagenase + MSAB group (Coln0.2 + MSAB group), 5 mg/kg/day MSAB (cat. no. HY-120697; MedChemExpress) was dissolved in an ultrasonic ice bath and administered intraperitoneally 2 h prior to each collagenase injection.

Techniques: Inhibition, Immunofluorescence, Staining, Marker, Western Blot

Journal: Journal of the American Society of Nephrology

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q10 Supplementation

doi: 10.1681/asn.2016060626

Figure Lengend Snippet: Figure 1. Hemolymph protein marker ANF-RFP and AgNO3 levels in nephrocytes expressing Coq-RNAi transgenes. (A) Fluorescence micrographs showing nephrocytes of adult flies 1-day postemergence. ANF-RFP fluorescence (red) is shown in the left panels. Right panels 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. Coq-IR flies carry Dot- Gal4 driving an RNAi transgene to silence expression of Coq2, Coq6, or Coq8 genes. (B) Quantification of nephrocyte RFP fluorescence, expressed relative to control value. For each genotype, 30 nephrocytes (six nephrocytes from each of five flies) were examined (*P,0.05). (C) Photomicrographs showing nephrocytes of third instar larvae reared on standard fly food supplemented with AgNO3. Control flies carry the Dot- Gal4 driver but no RNAi construct. Coq-IR flies carry Dot-Gal4 driving an RNAi transgene to silence expression of the indicated Coq gene. Scale bar, 20 microns. (D) Quantification of AgNO3, expressed relative to control value. For each genotype, 30 nephrocytes (six nephrocytes from each of five larvae) were examined (*P,0.05).

Article Snippet: The wild-type COQ2 cDNAwas obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: AAH08804).

Techniques: Marker, Expressing, Fluorescence, Control, Transgenic Assay, Construct

Journal: Journal of the American Society of Nephrology

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q10 Supplementation

doi: 10.1681/asn.2016060626

Figure Lengend Snippet: Figure 2. Coq2 gene silencing induced abnormal slit diaphragm localization and col- lapsed lacunar channels with multiple slit diaphragms (A) TEM showing normal (control) nephrocyte ultrastructure with slit diaphragms and lacunar channels uniformly spaced along the circumference of the cell. Slit diaphragms localized exclusively at the mouth of the channel. In Coq2-IR nephrocytes, channels appeared narrower (collapsed) and in- terchannel spacing was interrupted and irregular. Slit diaphragms occurred not only at the channel mouth but also ectopically along the interior channel membranes. Scale bar, 200 nm. (B) Higher magnification TEM comparing normal control and Coq2-IR slit di- aphragm and lacunar channel ultrastructure. Ectopic slit diaphragms arranged in ladder- like configuration at points of channel narrowing are indicated by arrows. Scale bar, 100 nm. (C) Quantitation of normally localized slit diaphragms in control versus Coq2-IR nephrocytes. Average number of slit diaphragms positioned at mouths of lacunar channels per 2000 nm of cell circumference (*P,0.05). (D) The average distance (in nm) between normally localized slit diaphragms in control versus Coq2-IR nephrocytes (*P,0.05). (E) Quantitation of ectopic slit diaphragms in control versus Coq2-IR nephrocytes. Average number of slit diaphragms positioned along interior channel membranes per 2000 nm of cell circumference (*P,0.05). (F) Quantitation of slit diaphragms (normally localized plus ectopic) in control versus Coq2-IR nephrocytes. Total number of slit diaphragms per 2000 nm of cell circumference (*P,0.05).

Article Snippet: The wild-type COQ2 cDNAwas obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: AAH08804).

Techniques: Control, Quantitation Assay

Journal: Journal of the American Society of Nephrology

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q10 Supplementation

doi: 10.1681/asn.2016060626

Figure Lengend Snippet: Figure 3. Coq2 gene silencing induced elevated numbers of autophagosomes and abnormal mitochondria in nephrocytes. (A) Upper panels: TEM showing normal mito- chondria (m) in control nephrocyte of a wild-type fly. In Coq2-IR nephrocytes the mito- chondria are more abundant and morphologically abnormal. Autophagosomes (ap) are in evidence. Scale bar, 300 nm. Lower panels: higher magnification electron micrographs comparing normal control and Coq2-IR nephrocyte mitochondria. In nephrocytes in which Coq2 expression was silenced the mitochondria exhibited shortened cristae and fewer inner mitochondrial membranes. Scale bar, 200 nm. (B) Quantitation of mito- chondria in control versus Coq2-IR nephrocytes. Average number of mitochondria per 4 mm2 area of cytoplasm (*P,0.05). (C) Percentage of mitochondria exhibiting abnormal morphology in control versus Coq2-IR nephrocytes (*P,0.05). (D) TEM showing mi- tochondria and autophagosomes in control nephrocyte of a wild-type fly. In Coq-IR nephrocytes, the number of autophagosomes was increased. Scale bar, 300 nm. (E) Quantitation of autophagosomes in control versus Coq2-IR nephrocytes. Average number of autophagosome per 4 mm2 area of cytoplasm (*P,0.05).

Article Snippet: The wild-type COQ2 cDNAwas obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: AAH08804).

Techniques: Control, Expressing, Quantitation Assay

Journal: Journal of the American Society of Nephrology

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q10 Supplementation

doi: 10.1681/asn.2016060626

Figure Lengend Snippet: Figure 4. Coq2 gene silencing induced autophagy and mitophagy. (A) Fluorescence micrographs showing nephrocytes of adult female flies 1-day postemergence, ex- pressing a dual-labeled GFP-Atg8-mCherry autophagy marker protein specifically in nephrocytes (UAS-GFP-Atg8-mCherry construct driven by Dot-Gal4). Dotted lines indicate nephrocyte cell boundary. Atg8 is present in autophagosomes and sub- sequently accumulates in autolysosomes. Fluorescence from the compartmentalized marker protein is punctate in appearance. Both GFP and mCherry fluorescence can be detected in autophagosomes. In the acidic lysosome lumen, GFP fluorescence undergoes rapid quenching whereas mCherry retains fluorescence activity. There- fore only very early autolysosomes will transiently exhibit GFP fluorescence. Be- cause Atg8 accumulates to high levels in autolysosomes, mCherry fluorescence is relatively stronger in this compartment. Upper panels show control nephrocytes (normal Coq2 gene expression) in which autophagy is essentially undetectable.

Article Snippet: The wild-type COQ2 cDNAwas obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: AAH08804).

Techniques: Fluorescence, Labeling, Marker, Construct, Activity Assay, Control, Gene Expression

Journal: Journal of the American Society of Nephrology

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q10 Supplementation

doi: 10.1681/asn.2016060626

Figure Lengend Snippet: Figure 5. Simultaneous silencing of Coq2 and autophagy genes induces synergistic effects. (A) Fluorescence micrographs showing nephrocytes of 1-day postemergence adult flies transgenic for Hand-GFP (GFP expressed under the control of a Hand gene enhancer to confirm pericardial nephrocyte cell identity).23 ANF-RFP (red) is merged with GFP (green, predominantly in nucleus). Control flies carry the Dot-Gal4 driver but no RNAi construct. Gene-IR flies carry Dot-Gal4 driving RNAi transgenes silencing endogenous Coq2, Atg1, or Atg6 genes singly or in combination. (B) Quantitation of nephrocyte RFP levels, relative to control. For each genotype, 30 nephrocytes (six nephrocytes from each of five flies) were examined (* indicates sig- nificance compared with control, # indicates significance compared with Coq2-IR; P,0.05). (C) Oxidized DHE fluorescence (red) in nephrocytes of third instar larvae. Dashed lines indicate cell nuclei (determined from DAPI staining, not shown). (D) Quantitation of DHE fluorescence intensity expressed relative to control nephrocytes. For each genotype, 30 nephrocytes (six nephrocytes from each of five larvae) were ex- amined (* indicates significance compared with control, # indicates significance com- pared with Coq2-IR; P,0.05).

Article Snippet: The wild-type COQ2 cDNAwas obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: AAH08804).

Techniques: Fluorescence, Transgenic Assay, Control, Construct, Quantitation Assay, Staining

Journal: Journal of the American Society of Nephrology

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q10 Supplementation

doi: 10.1681/asn.2016060626

Figure Lengend Snippet: 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 con- struct. Coq2 flies carry Dot-Gal4 driving RNAi transgene silencing Coq2 expression. (B) Quantitation of RFP levels in control versus Coq2-IR nephrocytes (expressed rel- ative 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).

Article Snippet: The wild-type COQ2 cDNAwas obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: AAH08804).

Techniques: Functional Assay, Fluorescence, Control, Transgenic Assay, Expressing, Quantitation Assay, Staining

Journal: Journal of the American Society of Nephrology

Article Title: A Personalized Model of COQ2 Nephropathy Rescued by the Wild-Type COQ2 Allele or Dietary Coenzyme Q10 Supplementation

doi: 10.1681/asn.2016060626

Figure Lengend Snippet: Figure 7. A normal allele of the human COQ2 gene but not a mutant allele (COQ2- S146A) rescued nephrocyte function in flies expressing Drosophila Coq2-IR. (A) Fluorescence micrographs showing uptake of ANF-RFP nephrocytes of 1-day post- emergence adult flies. Left panels (MHC-ANF-RFP) show intracellular ANF-RFP fluo- rescence (red). Right panels (MHC-ANF-RFP Hand-GFP) show RFP (red) merged with GFP (green, mostly nuclear). Hand-GFP expression confirms pericardial nephrocyte cell identity. All flies are transgenic for Hand-GFP. Control flies carry the Dot-Gal4 driver but no RNAi construct. Coq2-IR flies carry Dot-Gal4 driving RNAi transgene silencing the endogenous Drosophila Coq2 gene expression, and where indicated, also a UAS-COQ2 (wild-type human COQ2 allele) or a UAS-COQ2-S146N (mutant human COQ2 allele).6 (B) Quantitation of RFP levels (expressed relative to control) in control versus Coq2-IR nephrocytes expressing normal COQ2 or mutant COQ2- S146N. For each genotype, 30 nephrocytes (six nephrocytes from each of five flies) were examined (*P,0.05).

Article Snippet: The wild-type COQ2 cDNAwas obtained from OriGene, and encodes the common 371-aa isoform (GenBank ID: AAH08804).

Techniques: Mutagenesis, Expressing, Fluorescence, Transgenic Assay, Control, Construct, Gene Expression, Quantitation Assay

Journal:

Article Title: Identification of a Third msa Gene in Renibacterium salmoninarum and the Associated Virulence Phenotype

doi: 10.1128/AEM.70.11.6488-6494.2004

Figure Lengend Snippet: Distribution of the msa3 locus in 26 isolates of R. salmoninarum: Southern blot analysis of 500 ng of XhoI-digested genomic DNA with an msa ORF probe. Lane 1, Little Goose; lane 2, D6; lane 3, 91-127; lane 4, Bonneville-1; lane 5, Lookingglass; lane 6, Round Butte; lane 7, LR-95; lane 8, Marion Forks; lane 9, Lake Billy Chinook; lane 10, Bonneville-5; lane 11, MK91; lane 12, ss-ChS-94-1; lane 13, Cow-ChS-94; lane 14, W88B2; lane 15, GL64; lane 16, Sawtooth91; lane 17, DWK91; lane 18, DWK90; lane 19, MT239; lane 20, BPA2001-6031; lane 21, BPA2001-6050; lane 22, Lost18; lane 23, MMMvir; lane 24, ATCC 33209T; lane 25, Carson5b; lane 26, Willamette. Lanes m1 contained molecular mass marker II (Boehringer Mannheim), and lanes m2 contained molecular mass marker VII (Boehringer Mannheim). Sizes (in kilobases) are indicated next to the gels. Blot images were captured with an ImageStation 440CF by using the 1D Image Analysis software (version 3.5.3; Kodak). The ratios of densitometric measurements for the 8.4-kb band to densitometric measurements for the 6.4-kb band (msa3:msa1) and the ratios of densitometric measurements for the 2.7-kb band to densitometric measurements for the 6.4-kb band (msa2:msa1) are shown beneath the lanes.

Article Snippet: This possibility was tested by challenging juvenile chinook salmon ( O. tshawytscha ) with isolates bearing msa3 (MMMvir, Bonneville-1, and Carson 5b) or lacking msa3 (type strain ATCC 33209 T and Marion Forks).

Techniques: Southern Blot, Marker, Software

Journal:

Article Title: Identification of a Third msa Gene in Renibacterium salmoninarum and the Associated Virulence Phenotype

doi: 10.1128/AEM.70.11.6488-6494.2004

Figure Lengend Snippet: Median survival times and proportions of susceptible fish surviving at the end of the experimental challenges with five isolates of R. salmoninarum a

Article Snippet: This possibility was tested by challenging juvenile chinook salmon ( O. tshawytscha ) with isolates bearing msa3 (MMMvir, Bonneville-1, and Carson 5b) or lacking msa3 (type strain ATCC 33209 T and Marion Forks).

Techniques: