Sildenafil (C22H30N6O4S), 1 gram
One day before SCNT, donor cells were induced into quiescence via serum starvation (0.5 % FCS). On the day of SCNT, cells were trypsinized, washed, and resuspended in SOF-Hepes medium.
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Matured oocytes (with extruded polar body) were selected and zona pellucida was removed using 0.5 % pronase in DPBS.
| Property | Description | Value |
|---|---|---|
| Molecular Formula | - | C22H30N6O4S |
| Molecular Weight | - | 474.58 g/mol |
| Melting Point | - | 187°C |
| Solubility | In water | Very low |
| pKa | - | 6.8 |
All manipulations were performed in SOF-Hepes with 10 % FCS.
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Enucleated cytoplasts were exposed to phytohemagglutinin and fused with donor cells via double DC-pulse (1.2 kV/cm), then returned to maturation medium. As nuclear donor, we used a cell clone obtained by homologous direct recombination (HDR)11 that harbors a 106 bp deletion on the left homology arm of exon 3 as well as a puromycin-resistance cassette in both alleles of the SURF1 gene. Embryos were cultured in modified mSOF medium with amino acids and 4 mg/ml BSA, using a modified Well-of-the-Well system. Sows were synchronized with 12 mg/day of altrenogest for 15 days.
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Then, it was switched back to ACSF for 20-30 min until cellular calcium levels recovered to their original baseline. For each calcium signal, the area under the curve (AUC) and the peak amplitudes ΔF/F0 (in %) were analyzed. After washing with Tyrode’s solution, the cells were incubated for 10 min at room temperature (RT) in Tyrode’s solution to allow fura-2 de-esterification and equilibration. Next, the Tyrode’s solution was replaced by a calcium-free Tyrode’s solution (129 mM NaCl, 1 mM MgCl2, 5 mM KCl, 30 mM D-glucose, 25 mM HEPES, 0.5 mM EGTA, pH 7.4), and immediately placed in a temperature- and CO2-controlled (37 °C, 5 % CO2, and ∼20 % O2) sample holder attached to the stage of an Axio Observer 7 inverted microscope (Zeiss, Jena, Germany) equipped with a x 40 objective (F-Fluar M27; NA 1.3; oil immersion; Zeiss) and x 1 magnification changer (Tubelens Optovar; Zeiss). Fura-2 was alternatingly excited at 340 nm and 380 nm using a Sutter Lambda DG5 wavelength switcher (Sutter Instruments, Novato, CA, USA), FT 430 dichroic mirror, and BP525/50 emission filter.
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Fura-2 emission signals were captured in the dark using an Axiocam 702 camera (Zeiss), applying an exposure time of 20 ms and image acquisition interval of 1 s. Microscopy hardware was controlled by Zen Pro software (version 3.2; Zeiss). During time lapse recordings, 1 μM thapsigargin (TG) (Thermo Fisher Scientific) was added to the extracellular medium by gentle pipetting. Numerical fluorescence intensity data was extracted from the fura-2 images using FIJI software (version 1.53q; ROIs were manually defined for each time lapse recording in the cytosol of individual cells and extracellular background. Next, in each image fura-2 emission signals were quantified for multiple cells at both excitation wavelengths.
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All cytosolic ROIs were of identical size and remained inside the cytosol during the full duration of the recording. Cytosolic fura-2 signals were first individually background-corrected for each excitation wavelength, after which the ratio value (340/380 nm) was calculated. 10 μM ROCK inhibitor was added to the final media after splitting to promote neuron survival. Experiments and quantification were performed as previously described.81 Neurons generated for neurite outgrowth quantification were treated with 10 μM sildenafil in 0.1 % DMSO or 0.1 % DMSO alone following media changes on days 8, 10, and 12. On day 14, the neurons were split and seeded onto black-wall, flat-clear-bottom 96-well plates (Greiner) at a density of 5,000 cells/well, and treated once more with Sildenafil or DMSO. On day 13, they received 0.15 mg PgF2α, followed by 1000 IU hCG 96 h after the last altrenogest dose.
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On day 6 of SCNT embryo development (compacted morula to early blastocyst stage), embryos were surgically transferred via mid-ventral laparotomy, five days after standing estrus. Pregnancy was assessed via ultrasound on day 30 and confirmed on day 60 post-transfer.
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Neurons were then cultured until day 16, when they were fixed with PFA, blocked, and stained for TUJ1 and Hoechst. Neurite images were then acquired using the Operetta CLS high content imaging system (Revvity) the TUJ1 and Hoechst signals at 20 x. Following acquisition, the neurite images sildenafil citrate chewable tablets were analyzed using CellProfiler (version 4.2.5). Briefly, the Hoechst signal was used to identify the nucleus as a primary object and the TUJ1 signal was used to identify the neurites as secondary objects. The nuclear objects were then expanded to serve as a proxy for the cell body and subtracted from the TUJ1 signal to leaving only the individual neurites.
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The results were exported as CSV for data analysis. Mean neurite length was calculated as the mean over the median per experiment. On day 12, neurons were transfected with 10 nM of PRKG1 siRNA (Ambion) or scrambled siRNA (Ambion) by adding the transfection mix directly to the well. On day 14, after re-seeding the neurons onto the 96-well plates, neurons were transfected again with 10 nM of PRKG1 siRNA or scramble siRNA. On day 16, PRKG1 knock-down efficiency was evaluated by qPCR and neurite length was quantified with our HCA protocol.81 Sildenafil citrate (Sigma-Aldrich) was dissolved in DMSO, then diluted with tap water to obtain the final concentration of 400 mg/l drinking water and given ad libitum.
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Untreated mice (10 females, 8 males) were compared to sildenafil-treated Ndufs4 KO mice (6 females, 6 males). Electrocardiography (ECG) recordings were recorded from conscious mice, using the INDUS Rodent Surgical Monitoring system without anesthesia. Transthoracic echocardiograms to measure ventricular size, wall thickness, and ejection fraction were performed on mice using the Vevo 2,700 VisualSonics System. Intraperitoneal injection with 0.1 mg midazolam was applied for anxiolytic muscle relaxant effects, without any anesthetic agent that may cause cardiac suppression. Standard 2D, M-mode, Doppler, Tissue Doppler images were obtained using a 30-MHz linear array transducer, including the parasternal short and long axis. Farrowing was induced with PgF2α on days 114-115 of gestation. For body temperature detection we used a Thermal Imaging Camera (Dongguan Xintai Instrument Co., Ltd, China). SURF1 KO male piglets were treated immediately after birth with sildenafil citrate (n=3 animals with 2.1 mg/kg/day, n=4 animals with 0.5 mg/kg/day).
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For slicing, a Vibratome Microm HM 650 V (Thermo Fisher Scientific) was adjusted to the following settings: frequency of 60 Hz, amplitude of 1.0 mm, velocity of 33 mm/s, and slice thickness of 300 μm. The slices were directly transferred to a petri dish filled with HBSS, placed in a cell culture hood and washed four times. Finally, the slices were collected on Millicell-CM inserts (Millipore) and placed in a 6-well plate. 750 μl of cortical differentiation medium IV (CDMIV) were added in each well, so that the membrane was soaked but the top side of the slices was still exposed to air. cBOS were cultured at 36 °C and 5 % CO2 for around 30-59 days before starting calcium imaging experiments.
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For calcium imaging experiments,69 membrane permeable Oregon Green 488 BAPTA-1 AM (OGB-1, Invitrogen) was first solved in 20 % pluronic and 80 % DMSO and diluted to 200 μM in HEPES-buffered saline, which was then injected into cBOS and incubated for 30 minutes. During the incubation and throughout the experiments, cBOS were perfused with artificial cerebrospinal fluid (ACSF), containing 138 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 18 NaHCO3, and 10 glucose. The ACSF was bubbled with 95 % O2 and 5 % CO2, resulting in a pH of 7.4. OGB-1 was excited at 488 nm and signals were detected using the imaging software NIS-Elements and a variable scan digital imaging system (Nikon) attached to an upright microscope (Eclipse FN-1, Nikon). The microscope was equipped with a Fluor 40x/ 0.8 DIC M/N2 ∞ /0 WD 2.0 water immersion objective (Nikon) and an orca FLASH 4.0 LT camera (Hamamatsu Photonics).
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Images were routinely obtained at 1 Hz and emission was collected at >500 nm. Regions of interest (ROIs) representing cell somata were identified, and their signals were background-corrected. For calculating ΔF/F0, the traces were normalized to their initial baseline (first 30 s of measurement). Additionally, the fluorescence signals were corrected for bleaching and analysed with OriginPro Software (OriginLab Corporation). To probe for the cellular response to acute metabolic stress, ACSF was switched to glucose-free ACSF containing 5 mM sodium azide (Honeywell) and 2 mM 2-Deoxy-D-glucose (Apollo Scientific) for 2 min. At birth, the animals were separated from the sow and transferred to an incubator kept at 35 °C.
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All image analysis was performed by Vevo 2,100 analysis software (v.1.5) after rapid image collection. The diastolic function was measured using tissue Doppler imaging of the mitral annulus (E`) and conventional mitral inflow (E wave). Metabolic chamber by Promethion (Sable Systems International) was applied to characterize the metabolism of whole-animal energy expenditure.145 The 16-cage metabolic chamber system directly measures various parameters over a 72-hour period, such as heat production, oxygen consumption (VO2), CO2 production, and XY activity movement. The respiratory exchange ratio (RER) is calculated as the ratio of carbon dioxide production (VCO2) to oxygen consumption. This parameter is useful to estimate the metabolic status.
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The xy axis detection of animal motion measure activity during day-time and night-time. The formula applied was Heat = (1.232∗VCO2)+(3.815∗VO2). For generation of SURF1 KO pigs, we followed a protocol for somatic cell nuclear transfer (SCNT) based on our previous method11 with minor modifications. Briefly, ovaries from pubertal gilts of domestic pig, large white background were collected at a local abattoir and transported to the lab at 31-33 °C. Follicles larger than 3 mm were aspirated, and cumulus-oocyte complexes (COCs) were selected and matured in vitro at 38.5 °C under 5 % CO2 for 42 h.