Sympathetic neuropeptide Y has a protective effect on the cardiovascular system
Analysis of replicated and unreplicated experiments using graphPad (I): a new statistical technique for the statistical modelling of a cohort
All experiments were duplicated twice with the same conclusion, and in two independent cohorts as well. The figure legends state the exact numbers of replicated experiments. Data from the random allocation of samples and animals to experimental groups, and subsequent experiments, as well as blind and ad hoc registered data were collected and analysed.
GraphPad was used to perform analyses with one-way ANOVA and two-tailed unpaired Student t-tests.
Incubation of fat pads with iDISCO for 24 h staining, bleaching, disinfection and incubation
The fat pads were stained and cleared using a modified version of iDISCO. In brief, WAT or BAT was dissected from mice perfused with 20 ml of PBS, and the tissues were fixed with 4% PFA overnight. Tissues were pretreated once each with 20, 40, 60 and 80% ethanol and twice in 100% methanol for 30 min, all at room temperature, and then bleached with 5% H2O2 diluted in 100% methanol for 24 h at 4 °C with shaking. A small amount of antibiotics were used to rehydrate the samples and then in the buffer of a 2% Tween-20, 10 g heparin and 0.05% sodium azide. The sample was put to sleep at 37 C in a water bath where the permeabilizing solution and blocking buffer were used. For immunolabelling, samples were incubated for 5 days, with shaking, in primary antibodies diluted in antibody dilution buffer (5% DMSO, 5% goat serum, 0.2% Tween-20 and 10 μg ml−1 heparin in PBS) at 37 °C, washed with PTwH for 1 day and then incubated for a further 3 days with secondary antibodies and DAPI diluted using antibody dilution buffer at 37 °C, with shaking. The tissues were embedded in 1% agarose for one hour and then dehydrated in 20, 40, 60,80 and 100% methanol before being taken to a room temperature. Samples were incubated in dichloromethane (Sigma, catalogue no. 270997) until they sank and were then incubated in dibenzyl ether (Sigma, catalogue no. 179272) until clear. The samples were stored at room temperature, and transferred to ECi before they were displayed on a screen. Images of clear tissues were obtained using a microscope with a step size of 8 m, a light sheet thickness of 3.98 m and a horizontal dynamic focus set to eight steps. Samples were illuminated using a bidirectional light sheet and scanned under a ×2/0.5 NA objective with voxel size 4.03 μm (x), 4.03 μm (y) and 8 μm (z). A 488-nm laser with a 525/50 filter, a 561-nm laser with a 620/60 filter and a 638-nm laser with a 680/30 filter were used for AF488, AF546 and AF647, respectively.
There was a count of the percentages of PDGFR+ and DES+ cells. The percentages of NPY+ and TH+ neurons in the hypothalamus were manually counted. The views that were randomly picked and projected to the other side were used to calculate ganglionic mural cell coverage. Labelled areas were segmented using a threshold set by default, and coverage was calculated as area NPY+/area CD31+.
The percentage of EdU+ Cells was counted by using a method called detecting particles. The threshold was set using a method.
The outervation of NPY+ axons was quantified using the ‘Surface’ tool. Labelled areas of NPY+ axons and CD31+ vessels in whole cleared iWAT were automatically segmented, and the innervation of NPY in vasculature was calculated as volumeNPY+/volumeCD31+. The calculation for the coverage of mural cells was the same as for volumeDES+/volumeCD31+. Confocal images showing NPY+ innervation were quantified using Fiji with an automatic unbiased method. AreaNPY+ and areaCD31+ were segmented using the Otsu thresholding method and measured with the ‘measure’ program.
Using a plug-in, the overlapping between Th, NPY and CD31 was calculated. The regions that were randomly picked to make this calculation are , , and 30 . Labelled areas were automatically segmented using a threshold set by default, and overlapping percentages were calculated automatically by JACoP.
Source: Sympathetic neuropeptide Y protects from obesity by sustaining thermogenic fat
A dimensional reduction approach to gene expression data analysis and cell proliferation assay using the Click-iT EdU Cell Proliferation Kit (ThermoFisher)
Public scRNA-seq datasets were downloaded from Gene Expression Omnibus (GEO) and analysed using the following method. Cells with fewer than 200 unique detected genes or over 5% mitochondrial counts were discarded. The normalized gene x cell matrix was done in Seurat v.4.2.0. 50) in R (v.4.2.2). The data was scaled using the method of ‘ScaleData()’, with a linear dimensional reduction performed by the principal component analysis and calculation of UMAP coordinates. Cells were clustered using a technique called Findclusters which used dimensions of 15 and resolution of 0.5. Each cluster was identified based on differentially expressed genes and known markers in the published literature5,22,29,51,52,53.
The cell proliferation assay was performed with the Click-iT EdU Cell Proliferation Kit (ThermoFisher, catalogue no. C10340) for imaging. Mural cells were seeded at 0.5 × 105 per millilitre in 12-well plates on coverslips and cultured overnight before experiments. Cells were then labelled with EdU and cultured with or without 1 μM NPY or 2 μM For mural cells, the medium is for 6 h and has low-glucose DMEM with 2% FBS. Cells were fixed with 4% PFC and permeating buffer for 30 minutes and EdU was labelled using the Click-iT Plus reaction cocktail. The cells were imaged with a confocal microscope using Zen- black software and having a 20/ 0.8 NA objective. An area of 3,072 × 3,072 pixels was imaged for each biological replicate.
3T3-L1 cells were seeded in 12well plates at a density of one 105 per liter for the purpose of differentiating into white adipocytes. The medium was refreshed until cells were confluent. 2 days after cell confluence, the medium was put into each well, and 3 days later without or with 1 g of erythropoietin, in addition to the 500 M dexamethasone. Maintenance medium was refreshed every 2 days. 8 days was the length of time for each well to be differentiated. NPY treatments in experimental groups started when the induction medium was added and lasted throughout the whole differentiation process.
The mouse cell line was purchased without any testing for mycoplasma. The complete medium for cell culture includes high-glucose DMEM with glutamate (Sigma, catalogue no. 41965039) and 10% FBS (Sigma, catalogue no. 12133 C).
Cells were seeded at the indicated amount of 5 104 per deciL on glass coverslips and the indicated concentration ofPDGF-BB. After cell seeding, NPY and 220-BB were added to cells. Cells were collected 5 days later and either fixed with 4% PFA for imaging or lysed with Trizol for RNA extraction and qPCR.
There are three antibodies used to make fluorescent activated cell sorting and flow cytometry. Y 66 and anb185033 are examples. The FACS and flow cytometry were all killed with a very high amount of dilution of the antibodies. The LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (1:1,000, ThermoFisher, catalogue no. L10119) was used for live/dead staining.
During terminal blood collection, mice were euthanized by injecting 10 l g1 pentobarbital into their hearts and blood was collected from the left ventricle by a 25 G needle and syringe with 100 mM EDTA. Blood was then placed in tubes with 5 μl of 100 mM EDTA and centrifuged at 1,000× rcf at 4 °C for 15 min to separate plasma from blood cells, and then the supernatant was transferred to fresh tubes with DPP-IV inhibitor (final concentration 50 μM) and aprotinin (final concentration 500,000 IU ml−1). The concentration of NPY in mouse iWAT was determined using the NPY ELISA kit. EZRMNPY-27K). The data was recorded using a FLUOstar Omega microplate reader.
Proteins were extracted from iWAT as previously described48. The 50 M lysis buffer of the iWAT was filled with 700 l of homogenizing tubes. 500,000 IUml1 aprotinin is described in the catalogue no. Precellys 24 homogenizer was used for the homogenized A 6103-1MG. The clear portion of the lipids was retained after being removed from the tissue homogenize by centrifuging at 20,000 rcf for 15 minutes. The process was repeated a few times to make sure no more of the substance was left.
Source: Sympathetic neuropeptide Y protects from obesity by sustaining thermogenic fat
Comparison of the CFX 96 Real-Time PCR Detection System Gene Expression with Thermogenic and Adipogenic Genes
The data shown in Extended Data figs. 10m are qPCR data and were collected using the CFX 96 Real-Time PCR Detection System.
The ΔCt method was used to quantify gene expression using the following formula: relative expression = 2^ (−(Cttarget gene − Ctreference gene)). The thermogenic and adipogenic genes were compared by using the Ct method. 5b and extended data is included. 9l,m and 10m are included.
The following primary antibodies were used for immunofluorescent staining: rat anti-CD31 (BioLegend, catalogue no. 102501, MEC13.3, 1:100 dilution), rat anti-PLVAP (BioLegend, catalogue no. 120503, MECA32, 1:100 dilution), rabbit anti-DES (abcam, catalogue no. rabbit anti-NPY (Cell Signaling, catalog no. D7Y5A), 1:500 dilution. ab30914, 1:500 dilution), chicken anti-TH (Aves Labs, catalogue no. TYH73787982, 1:500 dilution), rabbit anti-TH (Sigma, catalogue no. The mouse anti-NPY1R was used in Santa Cruz. The rat has anti-PDGFR and the rabbit has anti-TAGLN. Cy3 anti-SMA (Sigma catalogue no. Ab 14106, 1:250 dilution). 1A4 is the goat anti-SOX17 and is listed in the R&D catalogue. The goat anti-EPHB4 is listed in the R&D catalogue. The anti-UCP1 and rabbit anti-UCP1 can be found in the catalogue.
Source: Sympathetic neuropeptide Y protects from obesity by sustaining thermogenic fat
Simultaneous imaging and microscopy analysis of white adipose tissue using coherent anti-Stokes Raman, two-photon excitation fluorescence and Zeiss inverted microscopes
Animals were anaesthetized with ketamine (100 mg kg−1) and xylazine (10 mg kg−1) and a small incision was made to expose epididymal white adipose tissue for microscopy analysis. Intravital images of space resolution 1,024 × 1,024 pixels from epididymal WAT were obtained simultaneously using an optical microscope with coherent anti-Stokes Raman scattering, two-photon excitation fluorescence and a bright-field microscope, with a confocal LSM 780-NLO Zeiss inverted microscope Axio Observer Z.1 (Carl Zeiss). All procedures were performed at the National Institute of Science and Technology on Photonics Applied to Cell Biology at the State University of Campinas. Adipocyte images from coherent anti-Stokes Raman scattering were acquired using two lines of lasers in wavelengths λpump = 803 nm and λStokes = 1,040 nm, and fluorescence images were acquired by two-photon excitation fluorescence using the exogenous fluorescence dye tetramethylrhodamine isothiocyanate dextran (Sigma, catalogue no. T 1162). In wavelength of Stokes is 1,040 nm. Following identification of blood vessels and acquisition of an initial image the video was started and, after ten frames, 50 µl (30 mg kg−1) of Dextran-70KDa (Sigma, catalogue no. T1162) was injected into the orbital plexus of the mouse. The video continued recording for approximately 12 min to capture vessel fluorescence and leakage. Fluorescence intensity over time was analysed for the whole duration of the video. A static image was analysed using a special tool. For evaluation of tissue leakage, a region of interest was selected inside the vessel and another in the tissue (outside the vessel). Fluorescence intensity was measured in both areas, and the intensity ratio between the tissue and within the vessel was calculated. Blood flow was calculated using the average of the raw intensity of dextran inside the artery over 100 s immediately following stabilization of the dextran signal after administration.
Images were acquired using a Zeiss LSM880 confocal microscope with Zen-black software (v.2.1). The samples were imaged with either a 10/0.45 NA objective or a 20/0.8 NA objective. Solid-state lasers of 405, 561 and 633 nm and an Argon 488 laser were used for DAPI, AF488, AF546 and AF647 fluorophores, respectively.
For staining, whole-mount, PFA-fixed ganglia were dehydrated once in each of 30, 50, 70 and 90% ethanol, twice in 100% ethanol—with shaking for 20 min at each concentration—and then rehydrated in 90, 70, 50 and 30% ethanol. Ganglia were then digested with 1.5 U ml−1 dispase-1 (Roche, catalogue no. 04942078001), 0.5 mg ml−1 collagenase (Sigma, catalogue no. C2674) and 300 μg ml−1 hyaluronidase (Sigma, catalogue no. H3884) diluted in PBS, with shaking at 37 °C in a water bath for 30 min. After blocking for 2 hours, the ganglia were put into a permeabilizing buffer and then incubated with primary neutralizing agents for 3 days. Ganglia were ready for further use three days later in the permeabilizing buffer at 4 C. Finally, ganglia were dehydrated once each in 30, 50, 70 and 90% ethanol and twice in 100% ethanol then cleared using ethyl cinnamate (ECi). There was a slide sandwich filled with ECi.
Fixed samples were processed in 70%, 80%, and 90%, and then in 50%, 100%, xylene, and 60 C of paraffin wax three times. Samples were embedded in paraffin wax, sectioned at 7 µm, mounted on coverslips and dried in an oven at 45–50 °C overnight. Before staining, samples were deparaffinized twice in 100 xylene for 5 min and then rehydrated twice in 100 xylene and then again in 95 and 80% water for 10 min. The antigen was retrieved by adding 0.05% trypsin to each slide for 20 minutes. After washing with running water, samples were stained using the same procedure as for fixed cells.
For cryosection, samples were first embedded in OCT (VWR, catalogue no. 361603E) and frozen at −20 °C until cryosectioning. The samples were mounted onto the SuperFrost slides with 10-m-thick sections. The same process as for fixed cells is used for staining.
Superior cervical ganglia and sympathetic axon bundles were dissected from adipose tissues of mice perfused with 20 ml of PBS, and the tissues were fixed in 4% paraformaldehyde (PFA, 043368.9 M, ThermoFisher) for at least 4 h. The cells are washed with PBS and then fixed using 4%pfa after at least 4 h. After stained at 4 C with primary markers dissolved in the permeabilizing buffer, secondary markers and DAPI were added to the bundle. As for immunofluorescence in fixed cells, cells were first incubated with the permeabilizing buffer for 1 h at room temperature and then stained overnight at 4 °C with primary antibodies diluted in the permeabilizing buffer. Samples were then washed and stained with DAPI and secondary antibodies for 1 h at room temperature. Sympathetic fibres or fixed cells were then mounted on slides with Fluoromount-G mounting medium (ThermoFisher, catalogue no. 00-4958-02).
A stereotaxic holder was used for the injections of mice that contained avertin. Either AAV2/9-hSyn-Cre-EGFP-WPRE-pA (Taitool, catalogue no. S0230-9, 2 × 1,012 viral genomes ml−1, 200 nl per side) or AAV2/9-hSyn-EGFP-WPRE-pA (catalogue no. The nucleus tractus solitarius has a dorsoventral 7.5, which is one of the reasons S0237-9 was injected into it.
Source: Sympathetic neuropeptide Y protects from obesity by sustaining thermogenic fat
Thermoimaging of Thcre and Cx3cr1GFP/+ Mice from Individual Cages for Fat-Induced Obesity
Animals aged 12–14 weeks were used for thermoimaging. The data was recorded using an Optris thermocamera and analysed with FOTRIC software. Animals were single housed and placed under the thermocameras, with their back shaved to expose the skin above iBAT. Animals were acclimatized for 4 days in individual cages with ad libitum access to food and water at room temperature under a 12/12 h light/dark cycle before a 14-h fast. The mice had access to water during their fast.
To record iBAT temperature, the camera manufacturer provided software that will store the temperature of the highest point in view. The iBAT temperature was taken as an average of temperatures over a 1-h period.
Animals aged 8–9 weeks were analysed for spontaneous locomotor activity (Panlab, Harvard Apparatus, LE001 PH Multitake Cage). Animals were kept in individual cages after a 12/12 h light/dark cycle with water and food supplied, and under controlled room temperature and humidity. Data was collected for 72 hours after a day of acclimatation. The activity was recorded using a piece of software. The MinispecLF50 was used to analyse the body composition of animals 7–9 weeks old.
ThCre mice (B6.Cg-7630403G23RikTg(Th-cre)1Tmd/J, stock no. 008601) and Cx3cr1GFP/+ mice (Cx3cr1tm1Litt/LittJ, stock no. 008451) were purchased from the Jackson Lab. Npyflox/flox mice were a donation from the I. Kalajzic Laboratory at the Department of Reconstructive Science, University of Connecticut44 under the material transfer agreement (MTA). The tissues of NPY-GFP mice were from the Brandy Memorial Laboratory at Yale University. M. Roberts at the University of Michigan gave the tissue from theRosa26tTomatod mice. NPy-cKO mice were created from crossed ThCre mice. Diet-Induced Obesity was achieved by feeding mice anHFD. Thisfeeding regime lasted for 10 weeks when they were 7 weeks old. The body weight of each mouse and food consumption in each cage were recorded weekly. All mice were group housed in standard housing under controlled room temperature (21–23 °C) and 50% humidity under a 12/12 h light/dark cycle and given access to diet and water ad libitum. All experimental procedures were performed on living animals in accordance with the UK ANIMALS ACTS 1986 under the project licence (PPL no. The UK Home Office granted personal licences. Ethical approval was provided by the Ethical Review Panel at the University of Oxford.
Using Imaris software to analyse embryos expressing major-satellite-mClover and H2B-mCherry
Experimental reproducibility was demonstrated as follows: Fig. 1g, two independent experiments; Fig. Two (2-cell), and three (1-cell, 4-cell) experiments are independent. There are two separate experiments in the picture. Two independent experiments are presented in fig. There are four independent experiments, two of which are 2- to-4-cell and 16-to-32-cell. 2i, two independent experiments, and 3a–d, two independent experiments.
To detect chromosome aberration using live imaging (Fig. 3c), we analysed images of embryos expressing major-satellite-mClover and H2B-mCherry using Imaris software (Bitplane). At an aphase timepoints, some bridges were identified with chromosome structures. We used images of embryos to analyse the foci at the M phase. To detect DNA repair foci, images at 6 min after nuclear envelope breakdown were processed using the 3D Spots detection function in Imaris software with a threshold of 2.0 times the cytoplasmic signal intensity. The detected spots were checked for quality manually and tracked over time through prometaphase. At each timepoint, the number of SLX4 foci on chromosomes was counted. To measure the cell cycle progression from the 1- to 8-cell stage (Fig. 4 and Extended Data Fig. 7), we analysed images of embryos expressing mEGFP–PCNA and H2B–mCherry with Imaris. To determine the timing of late S, images were processed using the ‘3D Spots’ function in Imaris with a threshold of 6.0 times the cytoplasmic intensity. The spots were manually checked for quality. The timing when the first PCNA spot appeared was defined by late S as the beginning of G2, whereas the last spot disappeared as the beginning of G2. We tracked all cells in an embryo while detecting chromosome bridges at each cell division. The error cells that exhibited chromosomes first for the first time during the 4-to-8- cell division were defined in Figure 4.
A/B compartment profile was computed from the.hic file using published mapped Hi-C datasets of sperm68 and cumulus60 cells. The lift over tool was used to change the genomic coordinates of PC1 profiles. 25% of PC1 values are divided into A1, A2, B2 and B1, each from the strongest A to the lowest B in the A/B compartment.
After linearization of the template plasmid, mRNA was synthesized using the mMESSAGE mMACHINE KIT (Ambion). When use is888-607-3166, the larger theRNAs, the longer they must be stored at 80 C. In vitro-transcribed mRNAs (0.9 pl of 150 ng µl−1 mEGFP-SLX4, 0.9 pl of 150 ng µl−1 mEGFP-PCNA, 0.9 pl of 150 ng µl−1 Major-satellite-mClover and 0.9 pl of 35 ng µl−1 H2B-mCherry) were microinjected into 1-cell embryos. Live-cell imaging was performed as previously described66 with some modifications. The Zeiss LSM780, LSM780 or LSM880 confocal microscope has a 40 C-Apochromat 1.2NA water-immersion objective lens and can be controlled by a multi-position autofocus macro67. For major-satellite imaging, 17 confocal z sections (every 1.5 µm) of 512 × 512 pixel xy images covering a total volume of 30.30 × 30.30 × 24.00 µm were acquired at 2 min 15 s intervals for at least 3 h just after nuclear envelope breakdown. For SLX4 it was possible to acquire 17 confocal parts every 2 m, covering a total of 30.30 30.30 32.00 m. For PCNA scanning, a total volume of 84.85 84.88 84.00 m was covered by the 29 confocal z sections. In Figs. The data is extended in a fig. During live monitoring, we chose and image blastomeres that were close to the objective lens, up to two blastomeres per embryo.
More than 30 embryos (C57BL/6 strain) at each developmental stage were collected. 1-, 2- and 4-cell embryos were collected at early S phase. Likewise, most 8-cell embryos were also collected at early-S phase (although some cells may not be at early S, as cells lose cell cycle synchrony after the 8-cell stage). Embryos were labelled with 100 μM 5-iodo-2′-deoxyuridine (IdU) for 30 min just before collection. After three quick washes with KSOM medium, the embryos were labelled with 100 μM 5-chloro-2′-deoxyuridine (CldU) for 30 min. The labelling reaction was stopped by washing the cells with ice-cold 1% FBS/PBS. The embryos were transferred into 1%PBS under the microscope with a mouth pipette. The embryos were then placed onto an APS-coated glass slide (Matsunami) with less than 1 μl of 1% FBS/PBS. Then, 20 μl of spreading buffer (0.5% SDS, 200 mM Tris-HCl (pH 7.5), 50 mM EDTA, 100 mM The NaCl that was added to the embryos on the glass slides was put into a petri dish, and it took 6 min for them to be fully formed. The slides were then gently tilted 20° from horizontal to stretch the DNA fibres. The DNA fibre slides were dried at room temperature for at least 1 h and the slides were fixed (methanol:acetic acid, 3:1) for 2 min. DNA fibres on the fixed slides were denatured with 1 M NaOH for 22 min, neutralized by five washes with PBS, and blocked with 1% BSA/PBST (0.05% Tween-20). The labelledDNA was detected using mouse anti-BrdU and rat anti-BrdU. After these antibody incubation steps for IdU/CldU detection, the slides were further incubated with mouse monoclonal antibody against ssDNA (1:100, Millipore, MAB3034, 16-19) for 30 min at 37 °C and Alexa Fluor 647 goat anti-mouse IgG2a secondary antibody (1:50, Invitrogen, A21241) for 30 min at 37 °C to avoid cross-reaction65. The samples were mounted with Prolong Diamond and photographs were taken with the DeltaVision Elite microscope. The oil-immersion objective is at 2,048 2,048. The emission band-pass filters that were used were 594 and , to avoid signal overlap. We figured out the extension rate to be around 4.3 kb per m using DNA as a control. The DeltaVision microscope was able to see the smallest single strands of DNA by using a maximum resolution of around 44 bp per min.
Images of DNA fibres were subjected to auto-thresholding with the ‘minimum’ or ‘default’ algorithm in Fiji software. Individual DNA fibres were identified by the ssDNA dots signals as being linearly arranged. The thresholded fibres containing the signals were automatically categorized into the groups with the subclasses that were mobile and intermediate. The immobile class forks were those with single dot signals of IdU + CldU and with gaps between Dots, reflecting extremely slow fork movement. There were gaps defined as those with at least one dot of the ssDNA signal. (immobile)). The mobile class forks were defined as those with a series of dot signals (≥2 dots) of IdU + CldU that contain a series of dot signals (≥2 dots) of CldU on either side of the same DNA fibre (Fig. 2h (mobile)). The intermediateclass forks were defined as those with an intermediate character between the other classes and also had single colour signals, as well as dot signals of IdU + chlU and with gaps between dots. The method for measuring the IOD on mobile fork class fibres is provided. 5b. The IOD between the immobile forks was calculated by measuring the distance between the brightest pixels in the centre of the dots using the Fiji software. To determine the fork speed of the mobile forks, CldU tracks flanked by IdU tracks were identified, their lengths were measured, and were divided by the duration of the second pulse (30 min). CldU tracks that had no ssDNA signals ahead of them were excluded from the fork speed measurement as these fibres may have been broken in the middle of the CldU track. The immobile fork speed measurement details are in Supplementary Note 2.
To quantify the levels of H3K9me2, p-Chk1 or γH2AX relative to the levels of histone H3, we obtained the mean signal intensity for H3K9me2, p-CHK1 or γH2AX within the nuclei (Ime_nuc). We took the Ime_nuc value from the mean signal intensity and subtracted it from the Ime_cyto. Similarly, we determined the histone H3 level within the same nuclei (IH3_nuc − IH3_cyto). The ratio between the two values is Ime_nuc Ime_cyto.
Embryos were fixed with 2% paraformaldehyde in PBS-polyvinyl alcohol (PVA) (pH 7.4) for 30 min. The embryos were washed multiple times in PBS-PHA-BSA, after being blocked and permeabilized by 1 mgml1 BSA and 0.1% Triton X-100. DNA was counterstained with 40 µg ml−1 of Hoechst 33342. The embryos were washed and transferred to a lab for more testing. The mouse anti-H2A.X wasphosphorylated by the Ser139, but the rabbit anti-histone H3 was not. The secondary antibodies were Alexa Fluor 488 goat anti-mouse IgG (H+L) (A11029); goat anti-rabbit IgG (H+L) (A11034); Alexa Fluor 555 goat anti-mouse IgG (H+L) (A21424) (1:400, Invitrogen).
Collection of single blastomeres was performed using micromanipulation. Five g of cytochalasin B was added to the M2 medium for 10 minutes. The zona pellucida was cut using a LYKOS laser system in a micromanipulation chamber, and then placed onto a warm stage in an inverted microscope. After cutting the hole of the zona pellucida, a fire polishing injection pipette (inner diameter 30 m) was used to isolated single blastomeres. For late-8-cell- and 16-cell-stage embryos, embryos were treated with TrypLE Express (Gibco) for 5 min before single-cell isolation. TE and ICM cells were isolated as previously described62. Microsurgical isolation of single cells was performed by micromanipulation. The cell was washed twice with PBS after single-cell isolation. The cell was moved to a tube with a sampling buffer.
SCNT was previously described. The groups of MII oocytes were transferred into droplets of M2 medium with 500 g cytochalasin B on the microscope stage. Oocytes undergoing microsurgery were held with a holding pipette. A hole was created in the zona pellucida through the use of an enucleation pipette. The enucleated oocytes were transferred into CZB after the MII spindle was in the pipette with a small volume of ooplasm. The donor cells were gently removed from the injection pipette after the nuclear injection. Each nucleus was injected into an enucleated oocyte, and these reconstructed oocytes were kept in the incubator until activation. Incubation in CZB with a combination of 10 mM SrCl2, 2 mMEGTA, and 5 m latrunculin A stimulated the creation of new oocytes.
All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation of the RIKEN Center for Biosystems Dynamics Research. Oocytes and sperm were produced from B6D2F1 and C57BL/6 mice. Oocytes and sperm were produced in C57BL/6 female mice and MSM/Ms male mice. Multiple mice were used to eliminate the effects of individual differences between mice. Figure 1b, 2 (4-cell), 4 (8-cell), 2 (16-cell) and 4 (ICM and TE) mice; Fig. 1d, 10 mice each (1-cell, 2-cell and 4-cell); Fig. 2d, 4 (1-cell), 3 (2-cell) and 3 (4-cell) mice; Fig. 2f, 3 (1-cell), 3 (2-cell) and 4 (4-cell and 8-cell) mice; Fig. There are 6 insitu and 2 invivo, with 8 and 7 in the lab and the other 4 in the real world. 3c, 15 mice each (2-to-4-cell, 4-to-8-cell and 8-to-16-cell); Fig. 3g, 3 mice each (2-to-4-cell, 4-to-8-cell, 8-to-16-cell and 16-to-32-cell); Fig. 3h, 4 (2-cell) and 3 (4-cell and 8-cell) mice; Fig. 3i, 5 (2-cell, 4-cell, and 8-cell) mice; Fig. 4d, 4 mice; Fig. 4h–i, 6 mice; Fig. 4j, 16 mice; Extended Data Fig. 6 are 2 to-4-cell cells, and 8 are two to-4-cell cells.
Cells were then labelled with EdU and cultured with or without 1M NPY or 2 M For mural cells, the medium is for 6 h and has low- Glucose DMEM with 2% FBS. The cells were imaged with a confocal microscope using Zen-Black software and having a 20/ 0.8 NA objective. The cells were collected 5 days later and either fixed with 4% PFA for imaging or lysed with Trizol for RNA extraction.