mu CT\boldsymbol{\mu C T} 分析--在新泽西癌症研究所临床前成像共享资源中心使用INVEON PET/CT(西门子医疗集团)对股骨和胫骨进行扫描。X射线管设置为80千伏和 500 muA500 \mu \mathrm{~A} ,以最高分辨率采集图像,不进行CCD分档,因此体素大小为 9.44 mum9.44 \mu \mathrm{~m} 。在 195^(@)195^{\circ} 角度范围内使用 0.66^(@)0.66^{\circ} 旋转步长,曝光时间为 6500 毫秒。在使用 INVEON Research Workplace 软件(西门子医疗集团)进行分析之前,用光束硬化校正和 Hounsfield 校正对图像进行重建。使用三维高斯滤波器进行处理以减少噪音后,人工分割出与皮质和骨小梁区域相对应的 ROI。
X 射线成像和骨溶解病变定量--通过 X 射线射线照相术评估小鼠的骨溶解病变。将麻醉小鼠放在独立包装的胶片(BIOMAX XAR 胶片,Cat#: F5763-50EA, Sigma-Aldrich)上,使用 MX-20 Faxitron 仪器在 35 千伏电压下照射 X 射线 15 秒。胶片用柯尼卡 SRX-101A 冲洗机冲洗。使用 Adobe Photoshop 软件(Adobe Systems Inc.
Western Blot 分析-SDS 裂解缓冲液(0.05 mM Tris-HCl、50 mM BME、2% SDS、0.1% 溴酚蓝、10% 甘油)用于收集细胞中的蛋白质。样品经加热变性后,在 10%SDS-page凝胶上进行等量加载、分离,然后转移到 PVDF 膜(Millipore)上,并用 5%牛奶进行阻断。用于免疫印迹的一抗包括:抗 beta\beta -肌动蛋白(1:10,000 稀释,Abcam,cat#ab6276,克隆 AC-15)、抗裂解 Caspase 3(1:1000 稀释,Cell Signaling,Cat# 9661S)、抗 IL6
结果以平均值 +-SD\pm \mathrm{SD} (标准偏差)或平均值 +-SE\pm \mathrm{SE} (平均值的标准误差)报告,如图例所示。统计比较采用非配对双侧学生 t 检验(不等方差假设)和 Mann-Whitney U 检验。所有体外实验重复三次,动物实验重复一次。所有实验的代表性图像(包括 Western 印迹和
免疫荧光)至少重复两次,并显示代表性图像(骨髓样本除外)。图 8 中的免疫荧光图像是同类骨髓细胞标本(治疗前与治疗后)的代表性图像。使用 ImageJ 软件对这些 IF 图像进行量化。
请使用后面的空表,按小标题定义的部分组织信息,跳过与您的研究无关的部分。请勿添加小标题。要添加一行,请将光标放在要添加行的上方行尾处,即表格右边界外。然后按 ENTER 键添加行。无需删除空行。每个条目必须在单独一行中;不要在一个表格单元格中列出多个项目。请参阅本文档末尾的示例表格,了解如何引用试剂。
关键资源表
试剂或资源
SOURCE
IDENTIFIER
抗体
碱性磷酸酶/ALP。
in 1:100, Rat
Alkaline Phosphatase/ALPL
in 1:100, Rat| Alkaline Phosphatase/ALPL |
| :--- |
| in 1:100, Rat |
研发系统
Cat# MAB29091 RRID:AB_11129451
β Actin [AC-15] in
1:10,000, 鼠标
beta Actin [AC-15] in
1:10,000, Mouse| beta Actin [AC-15] in |
| :--- |
| 1:10,000, Mouse |
Abcam
Cat# ab6276 RRID:AB_2223210
裂解的 Caspase-3(Asp175)
细胞信号技术
Cat# 9661 RRID:AB_2341188
1:1,000的GFP,鸡肉
Abcam
Cat# ab13970 RRID:AB_300798
Jagged1(H114) in 1:1,000,
Rabbit
Jagged1(H114) in 1:1,000,
Rabbit| Jagged1(H114) in 1:1,000, |
| :--- |
| Rabbit |
圣克鲁斯生物技术公司
Cat# sc-8303 RRID:AB_649685
兔 Jagged1,1:100(IF)
阿克里斯抗体
Cat# AP09127PU-N RRID:AB_2035312
1:100 的 Ki67,兔子
Abcam
Cat# ab15580 RRID:AB_443209
1:1,000的IL6,兔子
MBL 国际
Cat# JM-5144-100 RRID:AB_592035
1:1,000的PARP,兔子
细胞信号技术
Cat# 9542 RRID:AB_2160739
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
"Alkaline Phosphatase/ALPL
in 1:100, Rat" R & D Systems Cat# MAB29091 RRID:AB_11129451
"beta Actin [AC-15] in
1:10,000, Mouse" Abcam Cat# ab6276 RRID:AB_2223210
Cleaved Caspase-3 (Asp175) Cell Signaling Technology Cat# 9661 RRID:AB_2341188
GFP in 1:1,000, Chicken Abcam Cat# ab13970 RRID:AB_300798
"Jagged1(H114) in 1:1,000,
Rabbit" Santa Cruz Biotechnology Cat# sc-8303 RRID:AB_649685
Jagged1 in 1:100 (IF), Rabbit Acris Antibodies Cat# AP09127PU-N RRID:AB_2035312
Ki67 in 1:100, Rabbit Abcam Cat# ab15580 RRID:AB_443209
IL6 in 1:1,000, Rabbit MBL International Cat# JM-5144-100 RRID:AB_592035
PARP in 1:1,000, Rabbit Cell Signaling Technology Cat# 9542 RRID:AB_2160739| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
| :--- | :--- | :--- |
| Antibodies | | |
| Alkaline Phosphatase/ALPL <br> in 1:100, Rat | R & D Systems | Cat# MAB29091 RRID:AB_11129451 |
| beta Actin [AC-15] in <br> 1:10,000, Mouse | Abcam | Cat# ab6276 RRID:AB_2223210 |
| Cleaved Caspase-3 (Asp175) | Cell Signaling Technology | Cat# 9661 RRID:AB_2341188 |
| GFP in 1:1,000, Chicken | Abcam | Cat# ab13970 RRID:AB_300798 |
| Jagged1(H114) in 1:1,000, <br> Rabbit | Santa Cruz Biotechnology | Cat# sc-8303 RRID:AB_649685 |
| Jagged1 in 1:100 (IF), Rabbit | Acris Antibodies | Cat# AP09127PU-N RRID:AB_2035312 |
| Ki67 in 1:100, Rabbit | Abcam | Cat# ab15580 RRID:AB_443209 |
| IL6 in 1:1,000, Rabbit | MBL International | Cat# JM-5144-100 RRID:AB_592035 |
| PARP in 1:1,000, Rabbit | Cell Signaling Technology | Cat# 9542 RRID:AB_2160739 |
REAGENT or RESOURCE SOURCE IDENTIFIER
15D11 fully human antihuman Jagged1 antibody Amgen Inc. N/A
"Anti-Rabbit IgG -
Horseradish Peroxidase in 1:5,000, Goat" GE Healthcare Cat# RPN4301 RRID:AB_2650489
"Anti-Mouse IgG -
Horseradish Peroxidase in 1:5,000, Sheep" GE Healthcare Cat# NA931-1ml, RRID:AB_772210
Bacterial and Virus Strains
pMSCV-Puro Seth N., et al. 2011 NA
pMSCV-Jagged1 Seth N., et al. 2011 NA
pTyr-Co1a1-mJagged1 This paper NA
Biological Samples
14 pairs of bone marrow cytospin samples before and after Carboplatinum and paclitaxel treatment University Hospital Essen, Essen, Germany NA
Chemicals, Peptides, and Recombinant Proteins
OPG-Fc Amgen Inc. NA
Brefeldin A Sigma-Aldrich Cat# B6542-5MG
CCl (Carbon tetrachloride) Sigma-Aldrich Cat# 319961
Cisplatin Sigma-Aldrich Cat# 134357-100MG
Docetaxel Sigma-Aldrich Cat# 1224551-200MG
Hydrogen peroxide solution Sigma-Aldrich Cat# 216763
MRK003 Merck NA
N-Acetyl-L-cysteine Sigma-Aldrich Cat# A7250-5G
Paclitaxel Sigma-Aldrich Cat# T7191-25MG
(+)-Sodium L-ascorbate Sigma-Aldrich Cat# A4034
Tunicamycin Sigma-Aldrich Cat# T7765
Recombinant Mouse IL6 Protein R&D Biosystems Cat# 406-ML-005
Recombinant Mouse RANKL Protein R&D Biosystems Cat# 462-TEC-010
Critical Commercial Assays
Acid Phosphatase, Leukocyte (TRAP) Kit Sigma-Aldrich 387A-1KT
ALT Activity Assay Sigma-Aldrich MAK052
AST Activity Assay Sigma-Aldrich MAK055
Deposited Data
Gene expression microarray data GSE97997 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cpancgoafjcnxyv&acc=GSE979
Experimental Models: Cell Lines
Chinese hamster CHO ATCC Cat# CCL-61
Human embryonic kidney H29-Clone#7 Seth N., et al. 2011 NA| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
| :---: | :---: | :---: |
| 15D11 fully human antihuman Jagged1 antibody | Amgen Inc. | N/A |
| Anti-Rabbit IgG - <br> Horseradish Peroxidase in 1:5,000, Goat | GE Healthcare | Cat# RPN4301 RRID:AB_2650489 |
| Anti-Mouse IgG - <br> Horseradish Peroxidase in 1:5,000, Sheep | GE Healthcare | Cat# NA931-1ml, RRID:AB_772210 |
| Bacterial and Virus Strains | | |
| pMSCV-Puro | Seth N., et al. 2011 | NA |
| pMSCV-Jagged1 | Seth N., et al. 2011 | NA |
| pTyr-Co1a1-mJagged1 | This paper | NA |
| Biological Samples | | |
| 14 pairs of bone marrow cytospin samples before and after Carboplatinum and paclitaxel treatment | University Hospital Essen, Essen, Germany | NA |
| Chemicals, Peptides, and Recombinant Proteins | | |
| OPG-Fc | Amgen Inc. | NA |
| Brefeldin A | Sigma-Aldrich | Cat# B6542-5MG |
| CCl (Carbon tetrachloride) | Sigma-Aldrich | Cat# 319961 |
| Cisplatin | Sigma-Aldrich | Cat# 134357-100MG |
| Docetaxel | Sigma-Aldrich | Cat# 1224551-200MG |
| Hydrogen peroxide solution | Sigma-Aldrich | Cat# 216763 |
| MRK003 | Merck | NA |
| N-Acetyl-L-cysteine | Sigma-Aldrich | Cat# A7250-5G |
| Paclitaxel | Sigma-Aldrich | Cat# T7191-25MG |
| (+)-Sodium L-ascorbate | Sigma-Aldrich | Cat# A4034 |
| Tunicamycin | Sigma-Aldrich | Cat# T7765 |
| Recombinant Mouse IL6 Protein | R&D Biosystems | Cat# 406-ML-005 |
| Recombinant Mouse RANKL Protein | R&D Biosystems | Cat# 462-TEC-010 |
| Critical Commercial Assays | | |
| Acid Phosphatase, Leukocyte (TRAP) Kit | Sigma-Aldrich | 387A-1KT |
| ALT Activity Assay | Sigma-Aldrich | MAK052 |
| AST Activity Assay | Sigma-Aldrich | MAK055 |
| Deposited Data | | |
| Gene expression microarray data | GSE97997 | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cpancgoafjcnxyv&acc=GSE979 |
| Experimental Models: Cell Lines | | |
| Chinese hamster CHO | ATCC | Cat# CCL-61 |
| Human embryonic kidney H29-Clone#7 | Seth N., et al. 2011 | NA |
试剂或资源
SOURCE
IDENTIFIER
小鼠成骨细胞 MC3T3-E1 克隆 #4
ATCC
Cat# CRL-2393
小鼠间充质基质细胞(MSC)
Ren G. 等人,2008 年
NA
小鼠 PyMT-A-FIG
Wan, et al.
NA
人类乳腺癌 MCF7
ATCC
Cat# HTB-22
人类乳腺癌 SCP28
Kang Y., et al.
NA
人类乳腺癌 SCP2
Kang Y., et al.
NA
人类乳腺癌 SCP28Vector
Seth N., et al.
NA
人类乳腺癌 SCP28Jagged1
Seth N., et al.
NA
人类乳腺癌 SUM1315-M1B1
另一份手稿正在准备中,是 SUM1315 的衍生物
NA
人类胚胎肾脏 293T
ATCC
Cat# CRL-3216
小鼠乳腺癌 4T1.2
Eckhardt B.L. 等人,2005 年
RRID:CVCL_GR32
实验模型:生物体/菌株
小鼠 Bal/cJ
杰克逊实验室
Cat# 000651
小鼠 FVB/NJ
杰克逊实验室
编号 001800
小鼠 nu/nu
杰克逊实验室
Cat#: 002019
小鼠 Col1a1-Jag1 (FVB)
本文件
NA
XenoMouse XMG2KL
Kellermann S. 等人,2002 年
NA
XenoMouse XMG4KL
Kellermann S. 等人,2002 年
NA
寡核苷酸
实时 PCR 引物见表 S1
IDT
NA
Col1a1-Jag1-GT-F
IDT
CAACACCACGGAATTGTCAGT
Col1a1-Jag1-GT-R
IDT
GATGATGGGAACCCTGTCAA
软件和算法
AxioVision 软件版本 4.6.3
蔡司
NA
GeneSpring 13 软件
安捷伦
NA
ImageJ
国家卫生研究院
NA
INVEON 研究工作场所软件
西门子医疗
NA
NIS-Elements 共聚焦
尼康
NA
REAGENT or RESOURCE SOURCE IDENTIFIER
Mouse osteoblast MC3T3-E1 Clone #4 ATCC Cat# CRL-2393
Mouse mesenchymal stromal cell (MSC) Ren G., et al. 2008 NA
Mouse PyMT-A-FIG Wan., et al., 2014 NA
Human breast cancer MCF7 ATCC Cat# HTB-22
Human breast cancer SCP28 Kang Y., et al. 2003 NA
Human breast cancer SCP2 Kang Y., et al. 2003 NA
Human breast cancer SCP28Vector Seth N., et al. 2011 NA
Human breast cancer SCP28Jagged1 Seth N., et al. 2011 NA
Human breast cancer SUM1315-M1B1 another manuscript in preparation, derivative from SUM1315 NA
Human embryonic kidney 293T ATCC Cat# CRL-3216
Mouse breast cancer 4T1.2 Eckhardt B.L., et al. 2005 RRID:CVCL_GR32
Experimental Models: Organisms/Strains
Mouse Bal/cJ Jackson Laboratory Cat# 000651
Mouse FVB/NJ Jackson Laboratory Cat# 001800
Mouse nu/nu Jackson Laboratory Cat#: 002019
Mouse Col1a1-Jag1 (FVB) This paper NA
XenoMouse XMG2KL Kellermann S., et al. 2002 NA
XenoMouse XMG4KL Kellermann S., et al. 2002 NA
Oligonucleotides
See Table S1 for real-time PCR primers IDT NA
Col1a1-Jag1-GT-F IDT CAACACCACGGAATTGTCAGT
Col1a1-Jag1-GT-R IDT GATGATGGGAACCCTGTCAA
Software and Algorithms
AxioVision software version 4.6.3 Zeiss NA
GeneSpring 13 software Agilent NA
ImageJ National Institute of Health NA
INVEON Research Workplace software Siemens Healthcare NA
NIS-Elements Confocal Nikon NA| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
| :---: | :---: | :---: |
| Mouse osteoblast MC3T3-E1 Clone #4 | ATCC | Cat# CRL-2393 |
| Mouse mesenchymal stromal cell (MSC) | Ren G., et al. 2008 | NA |
| Mouse PyMT-A-FIG | Wan., et al., 2014 | NA |
| Human breast cancer MCF7 | ATCC | Cat# HTB-22 |
| Human breast cancer SCP28 | Kang Y., et al. 2003 | NA |
| Human breast cancer SCP2 | Kang Y., et al. 2003 | NA |
| Human breast cancer SCP28Vector | Seth N., et al. 2011 | NA |
| Human breast cancer SCP28Jagged1 | Seth N., et al. 2011 | NA |
| Human breast cancer SUM1315-M1B1 | another manuscript in preparation, derivative from SUM1315 | NA |
| Human embryonic kidney 293T | ATCC | Cat# CRL-3216 |
| Mouse breast cancer 4T1.2 | Eckhardt B.L., et al. 2005 | RRID:CVCL_GR32 |
| Experimental Models: Organisms/Strains | | |
| Mouse Bal/cJ | Jackson Laboratory | Cat# 000651 |
| Mouse FVB/NJ | Jackson Laboratory | Cat# 001800 |
| Mouse nu/nu | Jackson Laboratory | Cat#: 002019 |
| Mouse Col1a1-Jag1 (FVB) | This paper | NA |
| XenoMouse XMG2KL | Kellermann S., et al. 2002 | NA |
| XenoMouse XMG4KL | Kellermann S., et al. 2002 | NA |
| Oligonucleotides | | |
| See Table S1 for real-time PCR primers | IDT | NA |
| Col1a1-Jag1-GT-F | IDT | CAACACCACGGAATTGTCAGT |
| Col1a1-Jag1-GT-R | IDT | GATGATGGGAACCCTGTCAA |
| Software and Algorithms | | |
| AxioVision software version 4.6.3 | Zeiss | NA |
| GeneSpring 13 software | Agilent | NA |
| ImageJ | National Institute of Health | NA |
| INVEON Research Workplace software | Siemens Healthcare | NA |
| NIS-Elements Confocal | Nikon | NA |
我们感谢 N. Sethi、B. Ell、R. Chakrabarti、T. Celia-Terrassa、L. Wan、H.A. Smith、Z. Li、W. Lu 及其他实验室成员提供的技术支持和有益的讨论,感谢 J.J. Grady 和 C. DeCoste 在流式细胞仪方面提供的帮助,以及罗格斯新泽西癌症研究所 (RCINJ) 临床前成像设施的 T. Campbell 提供的 muCT\mu \mathrm{CT} 成像服务。我们感谢普林斯伯勒普林斯顿大学医疗中心的 M. Alpern 和 V. Buynevich 在血液样本分析方面提供的帮助。我们感谢安进发现研究部的主要科学家,特别是 L. Perkins 和 J. Ho,他们为 15D11 抗体的开发做出了贡献。这项研究得到了 RCINJ 研究发展奖、布鲁斯特基金会以及 METAvivor Research and Support (AWD1004691)、美国国防部 (BC123187)、美国国立卫生研究院 (R01CA134519 和 R01CA141062) 和安进公司对 Y.K. 的资助、本研究还得到了 RCINJ 的临床前成像设施和临床前成像与流式细胞术共享资源(P30CA072720)的支持。
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