Mice and Mice maintenance WT (C57BL/6J) and Ldlr−...

Mice and Mice maintenance
WT (C57BL/6J) and Ldlr−/− (B6.129S7-Ldlrtm1Her) 6-8 week old female mice were purchased from Jackson Laboratory. Jak2VF MxCre mice were created and reported previously.12 Bone Marrow (BM) from poly I:C treated female WT (C57BL/6J) or Jak2VF MxCre mice were transplanted into irradiated (10.5 Gy for 8.4 minutes) WT or Ldlr−/− recipients. WT BM recipients were fed a chow diet, while Ldlr−/− recipients were fed a Western diet (WD) (Harlan Teklad, TD88137) for the indicated period of time. Mice were housed under a 12 hr light /dark cycle with ad libitum access to water and food. All protocols were approved by the Institution Animal Care and Use Committee of Columbia University.
Atherosclerosis Lesion Analysis and Metabolic profiling
The heart with the aortic root attached was embedded in paraffin and then serially sectioned. The sections were stained with hematoxylin and eosin or trichrome (sigma, HT15) for morphometric lesion analysis. Five sections per mouse was used for total lesional and necrotic core area quatification as previously described.13 All these in vivo studies including the ones described below such as immunofluorescence and immunohistochemistry staining were performed with a blinding protocol. Each animal was assigned an arbitrary number and the data was collected based on the assigned number while the genotype of the mouse and experimental conditions were unknown to the data collector. Total plasma cholesterol and HDLcholesterol were measured using kits from WAKO diagnostics, total plasma triglyceride was measured using kit from Thermo (TR22421).
Immunofluorescence Staining
Paraffin-embedded slides were deparaffinized with Histo-clear and then rehydrated in deceasing concentration of ethanol. Identification of macrophages, neutrophils in atherosclerotic lesions were performed by immunostaining using anti-mouse CD107b (Mac-3) (BD 553322, 1˖100), anti-mouse Ly6G (Biolegend 127601, 1:200), anti-myeloperoxidase (R&D BAF3667, 1:30) Lesional MerTK was incubated with biotin-labeled MerTK (R&D system, BAF591) and Mac-3. The sections were incubated with primary antibodies overnight at 4˚C overnight then incubated with secondary antibodies for 30 min. For antibody specificity in immunofluorescence staining, isotype matched normal IgG was used as the control for each assay. Iron staining and TUNEL staining were performed using a commercially available kit (Abcam ab83366) and (Roche 12156792910). Images were acquired by using Leica immunofluorescence microscope.
Immunohistochemistry
Paraformaldehyde-fixed and paraffin-embedded lesions were deparaffinized and rehydrated, then incubated with anti-Mac-3 antibody (BD 553322, 1˖100) or with anti-Ter119 antibody (eBioscience 14-5921-82, 2 1:200) at 4˚C overnight and with secondary anti-Rat IgG (VECTOR, MP-7405) for 30 min. The reaction was developed with diaminobenzidine (DAB) staining (VECTOR, SK-4100). For antibody specificity, isotype matched normal IgG was used as the control for each assay.
Flow Cytometry
Flow cytometry to quantify peripheral blood neutrophils, monocytes, platelet-neutrophil aggregates, platelet-monocyte aggregates or bone marrow hematopoietic stem and progenitor cells profiles were performed as previously described.13 For analysis of erythrocytes from mice and human samples, the red blood cells were labeled with antibody against Ter119 (eBioscience 14-5921-82) or CD47 (BD 556045 for human and BD 563585 for mice) or Calreticulin (Abcam ab22683) in staining buffer (30 min, 4°C). For neutrophils activity assays, neutrophils were stained with antibodies against to CD45 (Biolegend 103133),
CD11b (Biolegend 101211), Ly6G (BD 561104) in staining buffer (20 min, 4°C). Formyl-Nle-Leu-PheNle-Tyr-Lys (FMLP) conjugated with Fluorescein (Thermofisher, 10 min, 4°C) was used to detect FPR1 expression. Flow cytometry was performed using the LSR Fortessa or LSRII (Beckton Dickinson) and data were analyzed using FlowJo software (Beckton Dickinson). Isotype matched normal IgG was used as the control in each flow cytometry assay.

Intravital Microscopy
Leukocyte-endothelial interactions along the carotid artery were analyzed by intravital epifluorescence microscopy. Mice were placed in supine position, and the right jugular vein was cannulated with a catheter for antibody injection. Intravital microscopy was performed after injection of antibodies to Ly6G (1μg; clone 1A8; eBioscience), Ly6C (1μg, HK1.4, Biolegend), and CD11b (1μg, M1/70, eBioscience) using an Olympus BX51 microscope equipped with a Hamamatsu 9100-02 EMCCD camera, and a 10× salineimmersion objective. Movies of 30s were acquired and analyzed offline. Rolling flux was assessed as cell moving across a line perpendicular to the carotid artery. Cells were considered adherent if not moving
during 30 seconds.
Flow Adhesion Assays
Flow adhesion assays were performed in vitro using IBIDI-Slide IV 0.1 flow chambers (Ibidi). Flow chambers were coated with 12 μg/mL of intercellular adhesion molecule-1 (ICAM1), vascular cell adhesion molecule-1 (VCAM1) and P-selectin (all from R&D systems). Neutrophils were stained with Ly6G (eBioscience 17-9668-82ˈ1:100) for 15 min on ice. After adding 1mM of Ca2+/Mg2+, 5x105 neutrophils were placed into flow chambers and flow was created during 3 minutes at 5μL/min at 37°C using a highprecision syringe pump. After perfusion with PFA 4%, cells were washed with PBS. Pictures were acquired using a climate chamber fluorescence microscope (Leica, DMi8) and adhered neutrophils were quantified using ImageJ software.
雷帕霉素/恩那度司他 atherosclerosis study
Chimeric Ldlr−/− mice with 20% Mx1-Jak2VF and 80% GFP bone marrow were administered WTD ENVIGO (TD.88137) supplemented with ruxolitinib (2 g/kg)35,36 for 12 weeks.

Bulk cell RNA-seq （做斑块/外周血的代谢、脂质组学等分析）
CD11b+ splenocytes were isolated from wild-type recipient mice 11 weeks after BMT. For hypercholesteraemia experiments, BMT was conducted in Ldlr−/− mice, four weeks after which a WTD was administered and sustained for seven weeks. Following WTD, mice were killed and CD11b+ splenocytes were collected and isolated into TRIzol reagent (Thermo Fisher Scientific). Aortic roots were also removed. RNA was isolated with RNeasy kits (Qiagen) and RNA-seq experiments were conducted as previously described37 on a NexSeq 500 (illumina). Genes were considered differentially expressed if they were significant at 5% false discovery rate (FDR) by DESeq2 and were at least twofold different in average reads per kilobase of transcript per million mapped reads (RPKM). Gene ontology analysis was conducted using the PANTHER database38. Extended Data Fig. 1 displays RNA-seq data. All data have been deposited into a public repository.
Aortic single-cell digest
Following termination of WTD, aortas were isolated, digested with 2.5 mg/ml liberase (Millipore Sigma, LIBTM-RO), 120 U/ml hyaluronidase (Millipore Sigma, H3506), and 160 U/ml DNase I (Millipore Sigma, DN25) for 45 min at 37 °C as previously described31. For qPCR analysis, cells were washed and stained with CD11b-APC, BV786-Ly6G, CD45.1-PE and CD45.2-BV421. Cells were sorted on a BD Influx sorter and isolated directly into TRIzol. qPCR analysis was conducted as described above.
Single-cell RNA-seq
For single-cell RNA-seq (scRNA-seq), after 12 weeks WTD, Ldlr−/− mice with control, Mx1-Jak2VF, or Mx1-Jak2VFGsdmd−/− bone marrow (not in a chimeric model) were killed, and aortas were digested as indicated above, except that necrosulfonamide (20 μM, Torcis) was supplemented in all media to prevent ex vivo necroptosis and gasdermin D oligomerization. Cell digests were sorted by flow cytometry for DAPI−CD45+ events, then run on Genomics scRNA-seq.
Western blot analysis（改为动脉粥样硬化斑块）
Lung tissue homogenate and macrophages were harvested, and proteins were extracted using RIPA buffer (Beyotime) containing protease inhibitors cocktail (Roche, Mannheim, Germany). To concentrate supernatants for western blot, 700 μL 100% methanol and 175 μL trichloromethane were added to 700 μL supernatant and vortexed for 30 s. Supernatants were then centrifuged at 13000 rpm for 5 min at 4°C. The supernatant liquid was removed, and added 700 μL 100% methanol, then centrifuged at 13000 rpm for 5 min at 4°C. Supernatants were discarded. And the remaining pellet was resuspended in 20 μL10% SDS, then added 4 μL 5×SDS -PAGE sample loading buffer (Beyotime) and boiled for 10 min at 95°C. The protein concentrations were measured with PierceTM Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific, Grand Island, USA). Equal amounts of protein or all protein from supernatants were subjected to 8%-12% gradient polyacrylic amide gel under reducing conditions. Gels were transferred into polyvinylidene difluoride membranes (Millipore, USA), blocked with 5% fat-free milk or 5% albumin from bovine serum (BSA, Biofroxx, Germany) at room temperature for 1.5 h. The blots were reacted with the primary antibody at 4 °C overnight, followed by horseradish peroxidaseconjugated secondary antibody (1:1000; Cell Signaling Technology, USA), and detection by ChemiDoc XRS (Bio-Rad, USA). The intensities of the bands were quantified using the Image Lab Analyzer software (Bio-Rad). β-actin, α-tubulin, or GAPDH were used as a loading control. The antibodies used in the study are shown in Table 1.
Real-time PCR （用动脉粥样硬化小鼠模型）
Total RNA was isolated from macrophages and lungs using RNAiso (TaKaRa Clontech, Japan). Reverse transcription with approximately 1 μg of total RNA was carried out in a T100TM Thermal Cycler (Bio-Rad, USA) using PrimeScriptTM RT reagent Kit (TaKaRa Clontech). Targeted gene expressions were measured by quantitative real-time PCR analyses using SYBR® Premix Ex TaqTM II system (TaKaRa Clontech) on a Bio-Rad real-time PCR system (CFX96 TouchTM; Bio-Rad, USA). The qPCR program was initiated at 95 °C for 30 s; 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. β-actin was used as an endogenous reference gene. The primers in Table 2 were purchased from Sangon Biotech (Shanghai, China). Gene expression abundance was calculated by the 2−ΔΔCt method.
Cytokine detection
IL-1β contents in the cell culture supernatant were measured after an additional 24 h of incubation using appropriate ELISA kits (Cat# TNF-α: 88-7324; IL-1β: 88-7013; Invitrogen, Thermo Fisher Scientific, USA).
我在做题为【Jak2V617f突变激活中性粒细胞mTOR–HIF-1α 通路，驱动脂质代谢重编程在动脉粥样硬化中的机制及干预研究】的研究，上述是我在文献中找到的实验方法描述（参数或细胞、通路等可能不一致），请根据主要内容及研究思路，写一份中文的研究方法，要求尽量详细，修正存在的错误，参考顶刊或国自然标书的实验设计方法，以及表达方式，保证逻辑的清晰，表达科学