Extended Experimental Procedures.
Zebrafish Strains, Mapping, and Genotyping
Following zebrafish lines were used for these studies: bars847, bart31131, wild-type AB/TAB, Tg (kdrl:GFP)s843, Tg (fli1a:EGFP)y1, Tg(Kdrl:mCherry)uto2
Bulked segregate analysis was performed as previously described (Michelmore et al., 1991
). Fine mapping of the t31131 mutation was performed with marker z22307 close to the mutation on contig: Zv8_scaffold 1100 of the zebrafish genome. Sequencing the coding region of the ubiad1
gene (ubiad1, LOC554810) revealed a t > a mutation at nucleotide 123 in bart31131
and a t > a mutation at nucleotide position 185 in bars847
Genotyping were performed by two sequential PCR, which products were analyzed by sequencing. We used following primers: first PCR: FW: 5′-CCTGTGTGTGTTGTGATCG-3′; RW: 5′-TAGGTGTTGACCAGGTTTCC-3′, second PCR: FW 5′-TGTAAAACGACGGCCAGTCTGGATGCAGGAGATGAAG-3′; RW: 5′- AGGAAACAGCTATGACCATCCAGCTTGTAGGCCAGAG-3′.
Morpholinos or mRNAs were injected at one cell-stage in different strains and phenotype was assayed between 48–72 hpf as described. Gene knockdown in zebrafish embryos was performed by microinjection of the following morpholinos: Control (5′-CCTCTTACCTCAGTTACAATTTATA-3′; 0.2 mM), ubiad1 (5′- GAAGCCAATCGGTATATTCACCTCC-3′; 0.2 mM), tnnt2a (5′- CATGTTTGCTCTGATCTGACACGCA-3′; 0.2 mM; Sehnert et al., 2002
), klf2a (5′-GGACCTGTCCAGTTCATCCTTCCAC-3′; 0.2 mM; Nicoli et al., 2010
), coq2 (5′-GTGTGAAATACAGAAAGCTCACCTA-3′; 0.2 mM), nos1 (5′- TTAATGACATCCCTCACCTCTCCAC-3′; 0.2 mM; North et al., 2009
). Morpholinos were synthetized from GeneTools and dissolved in nuclease-free water. Primers for testing ubiad1 morpholino were designed on zebrafish ubiad1 (GeneBank accession number: NM_001199726
) and are FW: 5′- CCGCAGGACGTGGTGATGTTTG-3′; REW: 5′- GTCTGAGTCCATGTCCCGCG-3′). Primers for testing coq2 morpholino were designed on zebrafish coq2 (GeneBank accession number: NM_001089486
) and are: FW: 5′-TTCAAAATATGTCACGGTGCT-3′; RW: 5′-GTCCTCGGGTCTGTTGATGT-3′. As control β-actin was detected with following primers: FW: 5′- GTATCCACGAGACCACCTTCA-3′; RW: 5′-GAGGAGGGCAAAGTGGTAAAC-3′.
Embryos from bar heterozygote intercrosses were injected at one-cell stage with 80pg of mRNA (mMessage Machine, Ambion) encoding for the indicated proteins. We also included a control mRNA for fluorescent protein H2B-cherry (50pg) in each injection.
Transplantation experiments were performed as previously described (Ho and Kane, 1990
). Donor embryos of the genotype Tg(fli1a:GFP)y1
were injected with 70 kDa Tetramethyl Rhodamine (TAMRA; Molecular Probes) at the one-cell stage and used for transplantations at the predome stage. Acceptor embryos were from wild-type or Tg(Kdrl:mCherry)uto2
lines as specifically indicated.
Generation of the Zebrafish Transgenic Line Tg(kdrl:Ubiad1-2A-mCherry)uto 35 and Analysis of Ubiad1 Endothelial Cell Autonomous Effect
The Tol2-based kdrl:Ubiad1-2A-mCherry construct has been assembled by using the Tol2 Kit and three-fragment gateway recombination cloning strategy (Kwan et al., 2007
). For 5′ entry cloning, the 6.4Kb Kdrl promoter was amplified from genomic DNA of wild-type zebrafish by PCR with following primers containing appropriate attB4 and attB1 sites (5′-GGGGACAACTTTGTATAGAAAAGTTGAAGCTTTTTCTTTTATTTAA-3′ and 5′-GGGGACTGCTTTTTTGTACAA ACTTGTTTGTCTGTTAAAATAACGT-3′, respectively). The PCR product was then cloned into pDONRP4-P1R by BP reaction to obtain p5E-Kdrl. For the middle entry cloning zebrafish ubiad1 was amplified with following primers containing appropriate attB1 and attB2 sites: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTAGGAGATGAAGCCGGCTGCGCTTTC-3′ and 5′- GGGGACCACTTTGTACAAGAAAGCTGGGTAAGAAATTCACAATAACGGCAGGCT-3′, respectively) and cloned into pDONR221 by BP reaction. The 3′ entry clone was p3E-2A-mCherrypA. Entry vectors were assembled together with the pDestTol2pA2 by LR reaction to create the expression vector pDestTol2-Kdrl-Ubiad1-2A-mCherry-pA. The pDestTol2-Kdrl-Ubiad1-2A-mCherrypA was mixed with mRNA for Tol2 transposase and microinjected into one-cell stage wild-type embryos. Injected embryos were raised to adulthood and founders were screened for red fluorescence in the vasculature. The transgene integration was also confirmed by PCR on genomic DNA with forward primer on ubiad1
sequence (5′- TGCACAGCAACAACACGGGG −3′) and reverse primer on mCherry sequence (5′- CGAACTCGTGCCGTTCACGG −3′). As control β-actin gene was detected with following primers: FW: 5′- GTATCCACGAGACCACCTTCA-3′; RW: 5′-GAGGAGGGCAAAGTGGTAAAC-3′. The transgenic fish line nomenclatures uto35
was approved by the Zebrafish Nomenclature Committee of ZFIN (http://zfin.org
). Microinjection of control and ubiad1 was performed as previously described in intercrossed from Tg(Kdrl:GFP)s843
and Tg(kdrl:Ubiad1-2A-cherry)uto 35
lines. After MO injection, double Tg[(Kdrl: GFP)s843:(kdrl:Ubiad1-2A-cherry)uto 35]
or single Tg(Kdrl: GFP)s843
were scored for embryos showing the barolo
phenotype as measurement of ubiad1
Whole-Mount In Situ Hybridization
Whole mount in situ hybridization was performed as previously described (Santoro et al., 2009
). The following oligonucleotides were used for antisense digoxigenin labeled probe: FW: 5′-CCGCAGGACGTGGTGATGTTTG-3′, RW: 5′- CGCGGGACATGGACTCAGAC-3′.
The TUNEL cell death assay was carried out using the “In Situ Cell Death Detection Kit” TMR Red (Roche) as previously reported (Santoro et al., 2007
). Zebrafish sections were analyzed by confocal microscopy. Acquisitions were quantitated with appropriate image processing tools as described in the “Image acquisition section.”
Western Blot Analyses on Zebrafish Embryos
Zebrafish embryos were collected and deyolked in a solution of PBS1X supplemented with complete protease inhibitors cocktail (Roche). For Ubiad1 detection, embryos were lysed in RIPA buffer. 50 μg of total extracts were not boiled and loaded on 12% SDS-PAGE denaturing gel. Western blot analyses were performed with mouse anti-UBIAD1 (9D4; 1:500).
Extraction of Single Cells from Zebrafish Embryos for Quantitation of Cellular ROS Level
embryos were dissociated into single cells by following procedure (modified from Covassin et al., 2006
). Embryos (n = 20) were collected and deyolked with forceps in sterile PBS supplemented with complete protease inhibitor cocktail (Roche). Embryos were then transferred into a 24 wells plate with 500 μl of Protease solution (0.25% trypsin, 1 mM EDTA, pH 8.0, sterile PBS) and 30 μl of Collagenase P (100 mg/ml, Roche). Embryos were incubated at 28°C for 30’ and homogenized every 10’with a 200 μl pipette tip. Lysis was blocked with 500 μl of FBS (Invitrogen) and cells were collected into a prechilled Eppendorf tubes and centrifugated at 450rcf / 4°C for 5′. Supernatant was removed and cells were washed with 1 ml of HBSS (GIBCO). Cells were then incubated with 1 ml of cold HBSS containing CellROX probe for 10’ and analyzed at appropriate fluorescence wavelength on FACSCalibur (Becton Dickinson) according to manufacture’s protocol. All procedures including the CellROX probe were carried out on dark.
Analysis of NADP/NADPH Ratio in Zebrafish Embryos
The NADP/NADPH ratio assay was performed on zebrafish tissue extracts using the NADP/NADPH assay kit (Abcam). Samples were extracted in 400 μl of the recommended extraction buffer and 50 μl were processed following instructions. OD450 measurements were made on a Glomax Multi Detection System (Promega) plate-reader. OD450 measurements were converted to nmol/sample using a standard curve and values were used for ratio as previously reported (O’Neill and Reddy, 2011
Chemicals for zebrafish treatments were dissolved in sterile water or dimethyl sulfoxide (DMSO). Zebrafish embryos were treated with the following drugs: Statins (Mevastatin, Simvastatin and Mevinolin, 5 or 500nM; Sigma), from 48 to 72 hpf or as otherwise indicated; Squalene Inhibitor (SQI, 10 μM; Calbiochem) from 32 hpf to 72 hpf, GGTI-2133 and FTI-277 (10–50 μM, Sigma) from 54 hpf to 72 hpf; Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) (500 μM, Sigma) from 32 to 65 hpf or as otherwise indicated, S-nitroso-N-acetyl-DL-penicillamine (SNAP) (100 μM, Sigma) from 48 hpf to 65 hpf.
Zebrafish and Human UBIAD1 Constructs
Zebrafish ubiad1 (GeneBank accession number: XM_681613.2
) and human UBIAD1 (GeneBank accession number NM_013319.2
) cDNAs were amplified using the following primers: FW: 5′-ATGAAGCCGGCTGCGCTTTC-3′; RW: 5′-TCACAATAACGGCAGGCT-3′; FW: 5′-ATGGCGGCCTCTCAGGTCCTG-3′; RW: 5′-TTAAATTTTGGGCAGACTGCCTGCTGG-3′ respectively and cloned in PCS2+ vector. All constructs were then amplified and subcloned into pGEM T-easy vector with following primers: human UBIAD1 forms:
FW: 5′-AAAGAATTCTCCATGGCGGCCTCTCAG-3′, RW: 5′-GAAAGATCTAATTTTGGGCAGACTGCC-3′, zebrafish ubiad1: FW: 5′ - AAAGAATTCGAGATGAAGCCGGCTGCG-3′, RW: 5′-TTTGGATCCCAATAACGGCAGGCTGCC-3′. Constructs were then cloned into pEGFP-N3 vector with restriction enzymes. All constructs were tested for comparable levels of expression and toxicity in cells.
Human SCCD Mutant UBIAD1 Constructs
Human SCCD mutant isoforms UBIAD1-N102S and UBIAD1-D112G were generated with QuickChange site-directed mutagenesis kit (Stratagene) with following primers: D112G FW: 5′- CTTTTCCAAGGGCATTGGCCACAAAAAGAGTGATG-3′, RW: 5′- CATCACTCTTTTTGTGGCCAATGCCCTTGGAAAAG-3′) and N102S: FW:5′-CGGGGCCGGTAATTTGGTCAGCACTTACTATGACTTTTCC-3′, RW:5′-GGAAAAGTCATAGTAAGTGCTGACCAAATTACCGGCCCCG-3′.
All constructs were then amplified and subcloned into pGEM T-easy vector with following primers: human UBIAD1 forms: FW: 5′-AAAGAATTCTCCATGGCGGCCTCTCAG-3′, RW: 5′-GAAAGATCTAATTTTGGGCAGACTGCC-3′, zebrafish ubiad1: FW: 5′ - AAAGAATTCGAGATGAAGCCGGCTGCG-3′, RW: 5′-TTTGGATCCCAATAACGGCAGGCTGCC-3′. Constructs were then sub-cloned into pCS2+ vector with specific restriction enzymes. All constructs were tested for comparable levels of expression and toxicity in cells.
Transfection of Human Primary Endothelial Cells
4.0 x105 human primary EC (P3-P4) were transfected with 50 or 100 pmoles of control-, UBIAD1-siRNA and COQ2-siRNA (siGENOME, Dharmacon) or 3 μg of expression constructs using Amaxa protocols (Nucleofector kit: VPB-1002, Nucleofector program: U-001). pCDNA-GFP (3 μg) was used as control of electroporation each time.
Subcellular Fractionation Experiments in Endothelial Cells
HUAEC (5 × 105
cells) were transfected with siRNA (60 pmoles) as previously stated. 2 × 107
cells were trypsinized and pelleted by centrifugation at 800rpm for 5′ and washed twice with cold PBS. Pellet was resuspended with mitochondria isolation reagent A (Mitochondria Isolation Kit Thermo Scientific), vortexed at 15Hz for 5 s and incubated on ice for 5 min. Cell suspension was then transferred to a prechill Dounce Grinder (Sigma) and homogenized on ice (30 strokes/3 times). Lysed cells were transferred to a new tube and mixed with 800 μl of mitochondria isolation reagent C (Mitochondria Isolation Kit; Thermo Scientific). Cell lysate was centrifuged at 700 g for 10 min at 4°C to separate membrane fractions (pellet) from mitochondria and cytosol (supernatant). Membrane fraction was then suspended in reagent A and centrifuged at 500 g for 10 min at 4°C (Lindner, 2001
). Pellet containing Golgi/ER/endosome fractions were then subjected to protein or lipid extractions as described. Supernatant containing mitochondria and cytosol fractions was transferred to a new tube and centrifuged at 3,000 g
for 15 min at 4°C. The upper phase was discarded (cytosol) and mitochondria pellet was washed twice with 500 μl reagent C and centrifuged at 12,000 g
for 5 min at 4°C (Rice and Lindsay, 1997
). Pellet containing mitochondria was subjected to protein or lipid extractions as described.
Western Blot Analyses on Human Endothelial Cells
For western blot analysis whole-cell extracts or subcellular fractions were lysed in RIPA Buffer and different amount of total protein or fractions were loaded on 4%–15% SDS-PAGE denaturing gel under reducing conditions. For Ubiad1 analyses samples were not boiled before loading on gel. Subsequent procedures were performed as described in Santoro et al., 2007
. Western blot analyses were performed with following primary antibodies: mouse anti-UBIAD1 (9D4; 1:500), goat anti TERE1 (UBIAD1)-N16 (1:250; SantaCruz), mouse anti βactin (1:5,000; Sigma), mouse anti Rab11 (1:1,000; BD Biosciences), rabbit anti HADHA (H
ehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA H
ydratase (trifunctional protein), A
lpha subunit) (1:5,000; Abcam).
UBIAD1 Monoclonal Antibody Production
Monoclonal antibody against UBIAD1 (clone 9D4) was produced in our laboratory by immunizing mice with recombinant GST-Ubiad1 (aa 96-126) fusion protein and its reactivity was characterized in ELISA, completion with peptide, western blot and immunofluorescence as described in the text.
UBIAD1 Localization in Endothelial Cells
For UBIAD1 localization studies human primary endothelial cells were transfected (where specified) and plated on fibronectin-coated glass slides. After 48 hr cells were fixed in 4% paraformaldehyde and incubated with the following primary antibodies: mouse anti UBIAD1 (1:5, 9D4), rabbit anti TGN46 (1:25, Abcam), mouse anti γ−Adaptin (1:100; BD Biosciences), mouse anti GM130 (1:100, BD Biosciences). Secondary antibodies were from Alexa Fluor (Invitrogen). Nuclei were stained with Hoechst. Localization studies were confirmed by treatment of cells with Brefeldin A (Sigma, 36μM) for 3h in cell culture medium.
Mitochondria Staining in Cultured Endothelial Cells
Mitochondria morphology was detected on human primary ECs by immunofluorescence with mouse anti-60 kDa mitochondrial protein antibody (1:100; Abcam). Hoechst was used for nuclei staining. ECs were then analyzed at confocal microscopy.
Samples (cells or zebrafish embryos) were lysed in 500μl water-ethanol 2:3 and a 5μl lysate aliquot was analyzed for protein content according to the Bradford assay. Lysates were extracted twice with 500μl of hexane and the resulting organic phase was evaporated dry under reduced pressure. The residue was dissolved in 200 μl of methanol, sonicated and lastly centrifuged (12,000 rpm, 5 min). The upper layer was then immediately subjected to HPLC analyses. A standard amount of CoQ6 (Spectra2000) was added to the cell or embryo lysates as an internal standard for the assessment of the recovery percentage. Quantitation of CoQ10, CoQ9, cholesterol, Vitamin K and CoQ10-13C6, CoQ9-13C6 analytes was done by external calibration curves and corrected to % recovery evaluated on CoQ6.
HPLC-UV analyses for CoQ10 and cholesterol from zebrafish embryo extracts were performed on a Waters Alliance System (separations module: 2695 model) equipped with a photodiode array detector (model 2998) and a Waters Symmetry C18
reverse phase column (4.6 mm × 75 mm, 3.5 μm); pure methanol was used for the separation of lipid extracts (flow rate: 1 ml/min; run time: 45 min). HPLC-MS analyses for CoQ10 and cholesterol from zebrafish embryo extracts were performed on a liquid-chromatography/electrospray ionization tandem mass spectrometry system (HPLC-ESI-MS, Waters 515 HPLC pump-3100 mass detector); 5 mM of ammonium formate was added to the eluent (100% methanol) to promote the ionization of the analytes (Ruiz-Jiménez et al., 2007
HPLC MS/MS analyses for Vitamin K2 from zebrafish embryo and cell extracts were done on API3200 HPLC-MS system equipped with APCI ion source (Applied Biosystems). Waters Symmetry C18 reverse phase column (4.6 mm × 75 mm, 3.5 μm particle size) was used with 100% methanol as the mobile phase (isocratic conditions, time course 60 min). Acquisitions were recorded in TIC (Total Ion Current) and SIM (Select Ion Monitoring) mode.
HPLC-MS analyses for CoQ10 and CoQ10-13C6 from cell extracts were done on an Ultimate 3000 HPLC (Dionex) coupled to a high resolving power mass spectrometer LTQ Orbitrap (Thermo Scientific), equipped with an atmospheric pressure interface and an ESI ion source. Samples were analyzed using an RP C4 column (Phenomenex Jupiter, 150 × 2 mm, 5 μm particle size, Phenomenex) at a 200 μL/min flow rate. The isocratic mobile phase composition was 95% of methanol and 5% of 10 mM aqueous formic acid. The injection volume was 20 μL. The tuning parameters adopted for the ESI source were: source voltage 4.5 kV, capillary voltage 48.00 V, and tube lens voltage 180 V. The heated capillary temperature was maintained at 265°C. The mass accuracy of the recorded ions (versus the calculated ones) was ± 5 mmu (milli-mass units). Analyses were run using both full MS (300–1,000 m/z range) and MS/MS acquisition in the positive ion mode. Analytes were quantified in the full MS mode as [M+Na]+. Five-points calibration curves were done by standard addition of the analytes on a cell lysate matrix in the range 0–500 ng/ml.
If not otherwise stated all chemicals used for extractions and HPLC analyses were from Sigma.
Exogenous CoQ10 and Vitamin K2 Administration
The composition of liposomes used for rescue tests was typically: 65% POPC (2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine), 5% DPPG (1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol), 25% cholesterol, 5% ubiquinone or menaquinone. A mixture of the appropriate amounts of phospholipids and cholesterol was dissolved in chloroform and the solution evaporated to dryness under reduced pressure (20 mbar) on a rotary evaporator (temperature of the bath 30°C). The lipid film was subsequently flowed with nitrogen for 2 hr to remove residual traces of organic solvents. The film was then heated to 65°C and hydrated with the dialysis buffer for 10 min. The resulting lipid suspension was sonicated two times for 20 s at a frequency of 20 kHz and finally dialysed overnight. The mean hydrodynamic diameter of the liposomes was determined by dynamic light scattering by means of a Malvern ZS Nanosizer (Malvern Instrumentation), while the efficiency of incorporation of ubiquinone or menaquinone into the liposomes was assessed by analytical HPLC-UV. Liposome suspensions typically contain 0.3-0.6 mM of CoQ10 or vitamin K2. Mitochondria-targeted CoQ10 (MitoQ) and relative control compound Decyl-TPP (D-TPP) were synthetized as previously reported (Murphy and Smith, 2007
) and dissolved in DMSO at appropriate working concentrations. Embryos were injected at one-single cell stage with the indicated amount of MitoQ and D-TPP. Primary endothelial cells (HUAEC) were silenced for appropriate gene and treated for 24 hr with MitoQ or D-TPP. Then, cells were analyzed by FACS analysis for mitochondrial membrane potential as previously described.
Mitochondrial Membrane Potential (ψ) Analyses
For mitochondria membrane potential analysis (ψ) 105 cells were incubated with JC-1 probe (Invitrogen, 4.5 μg/ml) for 15’ at 37°C/ 5% CO2. Then cells were washed twice with PBS1X, trypsinzed, collected on prechilled tubes and immediately analyzed on FACSCalibur (Beckton-Dickinson). All procedures were performed in the dark. JC1 stock solution (5 mg/ml) was diluted at intermediate stock solution (50 μg/ml) and vortex for several minutes to allow complete dissolution.
Lipid Peroxidation Analyses
Detection of lipid peroxidation was achieved in human primary endothelial cells (HUAEC) by measuring malondialdehyde-proteine (MDA) adducts with OxiSelect MDA Adduct ELISA Kit (CellBiolabs). 106 cells were trypsinized and centrifuged at room temperature for 800rpm /10’. Pellet was suspended with sterile PBS, supplemented with Complete Protease Inhibitor Cocktail (Roche) and lysed by micro-homogenization. Cell lysates were analyzed for protein content according to the Bradford assay and diluted as appropriate according to instructions. Enzyme reaction was stopped after 30’ and OD450 nm measurements were immediately read on Glomax Multi Detection System (Promega).
Quantitative Real-Time PCR Analyses
Total RNA was isolated with PureLink Micro-to-Midi Total RNA Purification System (Invitrogen) and cDNA was made with RT High Capacity kit (Applied Biosystems) according to the manufacturer’s protocol. Quantitative real-time RT-PCR on human cell samples was performed with ABI Prism 7300 real-time PCR System (Applied Biosystems) using Platinum Quantitative PCR SuperMix-UDG with ROX (Invitrogen). Following genes were analyzed with respective primers: human UBIAD1 (GeneBank accession number NM_013319.2
; FW: 809-832; RW: 874-895) and probe #36 (Roche); human COQ2 (GeneBank accession number NM_015697.6
; FW:937-959; RW: 1009-1030) and probe #37 (Roche). 18S rRNA was used as internal control.
Zebrafish genes for qRT-PCR analysis were: gpx1a
(GeneBank accession number: NM_001007281.2
) and hmox1
(GeneBank accession number: NM_001127516
). Genes were selected as reported to be upregulated under oxidative stress conditions (Malek et al., 2004
;) and qRT-PCR assay was performed by COGENTEC with following protocol: 5 ng of cDNA was amplified (in triplicate) in a reaction volume of 15 uL containing the following reagents: 7.5ul of TaqMan PCR Mastermix 2x No UNG (Life Technologies, Foster City, CA), 0.75 ul of TaqMan Gene expression assay 20x (Life Technologies, Foster City, CA). Real-time PCR was carried out on the 7500 Sequence Detector System (Life Technologies), using a pre-PCR step of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Preparations with RNA template without reverse transcriptase were used as negative controls. Samples were amplified with following Q-PCR assay (Life Technologies, Foster City, CA): gpx1a
: Dr03071768_m1; hmox1
: Dr03434096_m1; β-actin
: Dr03432610_m1. β-actin
gene was included as control housekeeping gene.
NOS Activity and NO Levels Analyses in Human Primary Endothelial Cells and in Zebrafish Embryos
The NOS enzymatic activity was quantified using NOS Activity Assay Kit (Cayman Chemicals; No.781001). The assay is based on the biochemical conversion of L-Arginine to L-Citrulline by the eNOS and nNOS enzymes. Samples were human primary endothelial cells (HUAEC) or zebrafish embryos (n = 100) at 72 hpf. Human primary endothelial cells (6
) were suspended with short treatment of trypsin and washed twice with PBS 1X. Cells were then centrifuged at 800 rpm for 10 min and suspended with 100 μl of Homogenization Buffer (Tris-HCl [pH 7.4]; 10 mM EDTA, 10 mM EGTA), microhomogenizated and sonicated. Samples lysates were centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was separated from pellet and quantified for proteins. Samples (50 μg) were incubated with a reaction mix containing 25 mM Tris-HCl [pH 7.4], 1 mM NADPH (Sigma, N1630), 0.6 mM calcium cloride, 0.1 μM calmodulin, 3 μM tetrahydrobiopterin (BH4), 1 μM flavin adenine mononucleotide, 1 μM flavin adenine mononucleotide and [3
H]-Arginine monohydrocloride (1 mCi/ml; Perkin Elmer, NET1123001MC). Samples were incubated at 37°C for 15 min and stopped by adding 400 μl of stop-buffer. [3
H]-Arginine was separated from [3
H]-Citrulline by passing samples through the equilibrated resin provided by the kit. Radioactivity was quantitated by liquid scintillation counting. Activities were calculated on percentage of Citrulline formed in the reaction in relation to total possible counts and expressed as arbitrary parameter (percentage of activity to control sample).
Live Tg(kdrl:mCherry)uto2 siblingss847 (sib) and bar
s847 (bar) mutant embryos were incubated at 56 hpf with 20 μM solution of DAF-2 DA (Calbiochem) in fish water, pH 7.0, in dark light at 28°C for 12 hr. After incubation, embryos were washed twice in fish water, pH 7.0, and analyzed at 72 hpf at stereo fluorescence microscopy.
All experiments were performed at least as three independent times for each conditions, and the error bars represent the mean of ± the standard error of the mean, unless otherwise stated. Statistical significance was performed by a Student’s test, as appropriate, and significance is reported in accordance with p value (
p ≤ 0.05,
p ≤ 0.01,
p ≤ 0.001).
Microscopy and Image Analysis
Images were acquired with TCSII SP5X confocal microscope, MZ16 FA stereomicroscope equipped with DCF300FY camera (Leica) or AZ100 stereomicroscope equipped with AxioCam MRm camera (Zeiss) and ApoTome AxioObserver Z1 (Zeiss). LAS AF and AxioVision software were used for analysis and image processing. Confocal acquisitions were analyzed for quantification of colocalization with ImageJ software applying Colocalization Finder PlugIn (French et al., 2008
Siblings and barolo
mutant embryos in Tg(kdrl:GFP)s843
background were embedded in 0.5% low melting agarose in presence of PTU in 8-well microscopic chambers (Ibidi) with glass bottom. Embryos were imaged with a 20x dry objective lens (Leica N/A 0.5) with a confocal scanning microscope Leica TCSII SP5. z Stacks were acquired at 512 × 512 resolution, scan speed of 400 Hz, and 3 micron z step size. Images were taken every 20 min for total movie duration of 12 hr. Videos were processed with ImageJ software (Abramoff et al., 2004
) and shown at 2 frames/s.