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78 protocols using nod cg prkdcscid il2rgtm1wjl szj mice

1

Lung-only Mice (LoM) Model for HCMV

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Lung-only mice (LoM) were generated as previously described (67 (link), 68 (link)). In brief, LoM were constructed by implanting of 2 pieces of human lung tissue (Advanced Bioscience Resources) subcutaneously into the back of male and female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice (NSG, The Jackson Laboratory). Expansion of the implants was monitored by palpation. Anesthetized mice were exposed to HCMV by direct injection of HCMV TB40/E (4.25 × 105 IU) into the implants in a total volume of 100 μL. Mice received vehicle control (0.5% methylcellulose, 0.5% Tween-80; p.o., b.i.d.), FLS-359 (50 mg/kg in vehicle; p.o., b.i.d.), or ganciclovir (100 mg/kg; i.p., daily) beginning 2 hours before infection. Human lung implants were harvested at 17 dpi and flash-frozen. Subsequently, implants were thawed and homogenized, and virus load was measured by TCID50 assay.
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2

Tracking Circulating Human Platelets

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Aliquots containing 1.108 washed human Cultured or Native platelets were injected through the retro-orbital vein into macrophage-depleted (by clodronate liposome abdominal injection on day −1), 7 to 8 week-old female NSG (NOD.Cg-Prkdc scid, Il2rg tm1Wjl/SzJ) mice (Jackson laboratory, Bar habor, USA). Circulating human and mouse platelets were analyzed by flow cytometry in whole blood samples drawn 3, 6, 15, 30, 120, 240, 1400, 2800 and 4320 min after transfusion. Human platelets were detected with a mAb (ALMA.17) against human GPIIb-IIIa. A mAb against GPIbβ (RAM.1) which reacts with human and mouse platelets was used to delineate the platelet region on the plots17 (link). The proportion of circulating Cultured or Native platelets recorded in the acquisition gate 3 min after transfusion was arbitrarily set to 1.
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3

Generation of Humanized and Murinized NSG Mice

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Female and male NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice from the Jackson Laboratories (Bar Harbor, ME, USA) or female Balb/c mice (BALB/cJRj, Janvier labs, Le Genest-Saint-Isle, France) were used for all experiments. Human CD34+ hematopoietic stem cells were isolated from human cord blood by magnetic separation (EasySep™ Human Cord Blood CD34 Positive Selection Kit II, STEMCELL technologies, Cologne, Germany) following the manufacturer’s protocol and purity controlled by flow cytometry. Humanized NSG (huNSG) mice were generated by engraftment of 100,000 hCD34+ cells (of a donor mix) at the age of 6 – 8 weeks, similar to the procedures described in earlier publications (15 (link)–18 (link)). Briefly, mice received whole-body irradiation with a sub-lethal dose of 2 Gy and hematopoietic stem cells were administered intravenously 2 hours later. For the generation of murinized NSG mice (muNSG), mice were treated as the huNSG mice, but received 100,000 bone marrow cells from a Balb/c donor instead of human CD34+ cells. The peripheral blood of huNSG mice was analyzed for the presence and frequency of murine CD45+, as well as human CD45+, CD3+ and CD20+ cells at week 18 post engraftment by flow cytometry.
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4

ReACp53 Suppresses Xenograft Tumor Growth

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All animal work was conducted in accordance with the NIH Guidelines of Care and Use of Laboratory Animals and approved by Duke Institutional Animal Care and Use Committee (IACUC/A092-16-04). Immunocompromised NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were from The Jackson Laboratories. 2×106 CWRR1 or C4–2 cells in 0.1ml 1× HBSS with 50% Matrigel (Corning) were inoculated subcutaneously into the right thigh of 6–8 weeks old male NSG mouse. When tumor volumes reached a size of 50–100 mm3 (approx. day 7 after inoculation), mice were randomly grouped into 3 groups (n=4) that received treatments of PBS (0.2ml), ReACp53 (15 mg/kg) or SCR (15 mg/kg) with intraperitoneal (IP) injections every 48 hours for two weeks. Tumors were measured every three days and tumor volumes were determined from caliper measurements of tumor length (L) and width (W) according to the formula (L×W2)/2.
Xenograft tumor were collected and paraffin-embedded for immunohistochemistry analysis.
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5

Comparative Reference Samples for Longitudinal Proteomics Workflow Monitoring

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As a quality control measure, two “Comparative Reference” (“CompRef”) samples were generated as previously described65 (link),66 (link) and used to monitor the longitudinal performance of the proteomics workflow throughout the course of this study. Briefly, patient-derived xenograft (PDX) tumors from established basal and luminal breast cancer intrinsic subtypes were raised subcutaneously in 8-week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (Jackson Laboratories, Bar Harbor, ME) using procedures reviewed and approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis. Xenografts were grown in multiple mice, pooled, and cryopulverized to provide a sufficient amount of uniform material for the duration of the study. Full proteome and phosphoproteome process replicates of each of the two CompRef samples were prepared and analyzed as standalone 10-plex TMT experiments alongside every four TMT-10 experiments of the study samples, using the same analysis protocol as the patient samples. These interstitially analyzed CompRef samples were evaluated for depth of proteome and phosphoproteome coverage and for consistency in quantitative comparison between the basal and luminal models (i.e., basal/luminal ratios across different replicates and batches; Figure S1C).
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6

Establishing Xenograft and Intracranial Glioblastoma Models

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Animal experiments were performed in accordance with a study protocol approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center. Xenografts were established in five-week-old male NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJmice (Jackson Laboratory) by injection of MT330 and SJG2 cells (1x106) directly into the flanks [37 (link)]. Tumors were measured weekly with a handheld caliper. In addition, luciferase-expressing GBM cells (106) were injected stereotactically into the superficial brain parenchyma of NSG mice through a burr hole in the skull as previously described [49 (link)]. NSG mice were injected with D-luciferin and subjected to live animal imaging weekly to quantify bioluminescence [38 (link), 49 (link)].
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7

ReACp53 Suppresses Xenograft Tumor Growth

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All animal work was conducted in accordance with the NIH Guidelines of Care and Use of Laboratory Animals and approved by Duke Institutional Animal Care and Use Committee (IACUC/A092-16-04). Immunocompromised NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were from The Jackson Laboratories. 2×106 CWRR1 or C4–2 cells in 0.1ml 1× HBSS with 50% Matrigel (Corning) were inoculated subcutaneously into the right thigh of 6–8 weeks old male NSG mouse. When tumor volumes reached a size of 50–100 mm3 (approx. day 7 after inoculation), mice were randomly grouped into 3 groups (n=4) that received treatments of PBS (0.2ml), ReACp53 (15 mg/kg) or SCR (15 mg/kg) with intraperitoneal (IP) injections every 48 hours for two weeks. Tumors were measured every three days and tumor volumes were determined from caliper measurements of tumor length (L) and width (W) according to the formula (L×W2)/2.
Xenograft tumor were collected and paraffin-embedded for immunohistochemistry analysis.
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8

Comparative Reference Samples for Proteomics

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As a quality control measure, two ‘‘Comparative Reference’’ (‘‘CompRef’’) samples were generated as previously described65 (link),66 (link) and used to monitor the longitudinal performance of the proteomics workflow throughout the course of this study. Briefly, patient-derived xenograft (PDX) tumors from established basal and luminal breast cancer intrinsic subtypes were raised subcutaneously in 8-week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (Jackson Laboratories, Bar Harbor, ME) using procedures reviewed and approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis. Xenografts were grown in multiple mice, pooled, and cryopulverized to provide a sufficient amount of uniform material for the duration of the study. Full proteome and phosphoproteome process replicates of each of the two CompRef samples were prepared and analyzed as standalone 10-plex TMT experiments alongside every four TMT-10 experiments of the study samples, using the same analysis protocol as the patient samples. These interstitially analyzed CompRef samples were evaluated for depth of proteome and phosphoproteome coverage and for consistency in quantitative comparison between the basal and luminal models (i.e., basal/luminal ratios across different replicates and batches; Figure S1C).
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9

Longitudinal Proteomics Workflow Validation

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As a quality control measure, two “Comparative Reference” (“CompRef’’) samples were generated as previously described (Li et al., 2013 (link); Tabb et al., 2016 (link)) and used to monitor the longitudinal performance of the proteomics workflow throughout the course of this study. Briefly, patient-derived xenograft (PDX) tumors from established basal and luminal breast cancer intrinsic subtypes were raised subcutaneously in 8-week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (Jackson Laboratories, Bar Harbor, ME) using procedures reviewed and approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis. Xenografts were grown in multiple mice, pooled, and cryopulverized to provide a sufficient amount of uniform material for the duration of the study. Full proteome, phosphoproteome and acetylome process replicates of each of the two CompRef samples were prepared and analyzed as standalone 10-plex TMT experiments alongside every 4 TMT-10 experiments of the study samples, using the same analysis protocol as the patient samples. These interstitially analyzed CompRef samples were evaluated for depth of proteome, phosphoproteome, and acetylome coverage and for consistency in quantitative comparison between the basal and luminal models.
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10

Basal and Luminal Breast Cancer Xenograft Proteomic Analysis

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Aliquots of frozen, powderized patient-derived xenograft tumors from established basal (WHIM2, passage 32) and luminal A (WHIM16, passage 33) breast cancer subtypes were obtained from the Washington University Clinical Proteomic Tumor Analysis Consortium (CPTAC) Proteome Characterization Center. The xenografts were raised subcutaneously in 8 week old NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice (Jackson Labs, Bar Harbor, Maine) as previously described [29 (link), 45 (link)]. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis, MO.
Two aliquots of xenograft tumor tissue from basal subtype and two aliquots of xenograft tumor tissue from luminal subtype were used. Tissues were sonicated in denaturing buffer (8 M urea, 50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 50 μM PR-619, 1 mM 2-chloroacetamide) supplemented with protease inhibitors (2 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM PMSF). One hundred μL denaturing buffer was added per 10 mg tissue. Lysates were sonicated and cleared by centrifugation at 16,000 x g for 20 min at 4 °C.
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