Journal: Human Reproduction (Oxford, England)

Article Title: Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys

doi: 10.1093/humrep/dex338

Figure Lengend Snippet: Photomicrographs illustrating immunohistochemical staining for estrogen receptor 1 (ESR1), progesterone receptor (PGR) MMP26, TIMP3, Ki-67 and androgen receptor (AR) in the endometrial functionalis zone of the macaque uterus from representative females in each treatment group (C, T, WSD, T+WSD). Brown staining denotes positive expression of proteins. Sections are counterstained with hematoxylin (blue) staining. ESR1, PGR, Ki-67 and AR staining is nuclear, while MMP26 and TIMP3 show cytoplasmic localization. Inset shows a negative control with an irrelevant antibody (Anti-Br(d)U). TIMP3 staining was localized to the predecidual cells around the spiral arteries.

Article Snippet: Antibodies used were against estrogen receptor 1 (ESR1,ER-ID5; Cat#: MS-354-P, Thermo Fisher Scientific), progesterone receptor (PGR, Cat#: Ms-298-P, 1 μg, Lab Vision/NeoMarkers, Fremont, CA, USA), androgen receptor (AR-F39 (Cat#: MU256, 1/50, BioGenex, Fremont, CA, USA), Ki67 (Cat#: MU370-UC, 1/200, BioGenex), MMP26 (Cat# ab57636; Abcam, Cambridge, MA, USA) and TIMP3 (Cat#:MAB973, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Immunohistochemical staining, Staining, Expressing, Negative Control

Journal: Human Reproduction (Oxford, England)

Article Title: Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys

doi: 10.1093/humrep/dex338

Figure Lengend Snippet: Expression of ESR1, PGR, MMP26 and TIMP3 mRNAs as detected by qRT-PCR in endometrial biopsies collected from macaques in the mid-luteal phase of the cycle. Significant (P < 0.05) differences between individual treatment groups are denoted by different uppercase letters above columns.

Article Snippet: Antibodies used were against estrogen receptor 1 (ESR1,ER-ID5; Cat#: MS-354-P, Thermo Fisher Scientific), progesterone receptor (PGR, Cat#: Ms-298-P, 1 μg, Lab Vision/NeoMarkers, Fremont, CA, USA), androgen receptor (AR-F39 (Cat#: MU256, 1/50, BioGenex, Fremont, CA, USA), Ki67 (Cat#: MU370-UC, 1/200, BioGenex), MMP26 (Cat# ab57636; Abcam, Cambridge, MA, USA) and TIMP3 (Cat#:MAB973, R&D Systems, Inc. Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Microenvironment

Article Title: Mammographic Density and Matrix Metalloproteinases in Breast Tissue

doi: 10.1007/s12307-009-0031-x

Figure Lengend Snippet: Proportion of subjects with positive stains for MMPs and TIMP3 by ethnicity

Article Snippet: Following TMA construction, 5 μm sections of TMA blocks were sent to Wake Forest University for immunohistochemical staining with the following markers: MMP1 (1906-1), MMP3 (1908-1), MMP9 (1939-1), MMP12 (1906-1; all Epitomics, Inc., Burlingame, CA), and TIMP3 (CA 0577; Cell Applications, Inc., San Diego, CA).

Techniques:

Journal: Cancer Microenvironment

Article Title: Mammographic Density and Matrix Metalloproteinases in Breast Tissue

doi: 10.1007/s12307-009-0031-x

Figure Lengend Snippet: Percent density for MMP1, MMP12, and TIMP3 by tissue type and ethnicity (Adjusted for age at mammogram, BMI, ethnicity, parity, age at first live birth, age at menarche, menopausal status, hormone use at mammogram, family history of breast cancer, and tumor stage)

Article Snippet: Following TMA construction, 5 μm sections of TMA blocks were sent to Wake Forest University for immunohistochemical staining with the following markers: MMP1 (1906-1), MMP3 (1908-1), MMP9 (1939-1), MMP12 (1906-1; all Epitomics, Inc., Burlingame, CA), and TIMP3 (CA 0577; Cell Applications, Inc., San Diego, CA).

Techniques:

Journal: Cancer gene therapy

Article Title: Tissue inhibitor of metalloproteinases-3 transfer suppresses malignant behaviors of colorectal cancer cells.

doi: 10.1038/cgt.2012.70

Figure Lengend Snippet: Figure 1. TIMP3 was downregulated in colon cancer tissues. (a) TIMP3 expression at mRNA level was detected in colon cancers and paired normal mucosal tissues. TIMP3 was expressed in normal mucosal tissues but not cancer tissues. (N, normal mucosal tissue and C, colon cancer tissue); (b) Western blot showed lower expression of TIMP3 in colon cancer tissues than in normal mucosa; (c) A pair of the stained tissues from the same patient to show TIMP3 expression (left: normal mucosal tissue, right: colon cancer tissue). TIMP3 expression was obvious in cytoplasm of normal mucosal cell, but was shut off in cancer tissue. (d) The quantitative analysis of the TIMP3 staining in tissue array. TIMP3 expression was much lower in cancer tissues than that in normal control tissues (compared with normal mucosa, *Po0.01).

Article Snippet: The TIMP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Staining, Control

Journal: Cancer gene therapy

Article Title: Tissue inhibitor of metalloproteinases-3 transfer suppresses malignant behaviors of colorectal cancer cells.

doi: 10.1038/cgt.2012.70

Figure Lengend Snippet: Figure 2. Ad-TIMP3 induced cell apoptosis and suppressed cell growth. (a) TIMP3 mRNA expression was gradually upregulated with increased MOI after Ad-TIMP3 infection, whereas TIMP3 was not detectable by RT-PCR in control CT26 cells. The fold change of TIMP3 mRNA expression relative to that of 50 MOI infection was calculated. (b) By Ad-TIMP3 infection, TIMP3 protein was increased at a dose-dependent manner. To show the exogenous TIMP3-induced apoptosis, we detected the PARP cleavage. It showed the PARP cleavage occurred with the intensity of Ad-TIMP3 infection. (c) CT26 cells were transfected by Ad-TIMP3 or Ad-Null at the indicated MOI for 72 h and then subjected to annexin V-FITC assay. Percentage of apoptosis was defined by % of cells that were FITC þ/PI and FITC þ/PI þ. The experiment was performed twice independently; (d) CT26 cells were infected with Ad-TIMP3 or Ad-Null at the indicated MOI for 72 h. Trypan blue exclusion was used to determine cell number (compared with Ad-Null, *Po0.05). (e) Ad-TIMP3 infection suppressed the activity of MMP2 and MMP9 in the culture supernatant. In the gelatin zymography assay, MMP2 and MMP9 activity was suppressed in a dose-dependent manner by Ad-TIMP3.

Article Snippet: The TIMP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Activity Assay, Zymography Assay

Journal: Cancer gene therapy

Article Title: Tissue inhibitor of metalloproteinases-3 transfer suppresses malignant behaviors of colorectal cancer cells.

doi: 10.1038/cgt.2012.70

Figure Lengend Snippet: Figure 3. Ad-TIMP3 impaired metastasis ability of cancer cells. (a) Adhesion ability of cancer cells was impaired by Ad-TIMP3 infection in a dose-dependent manner. The control virus Ad-Null did not significantly affect the adhesion ability at 50 MOI of infection. Similarly, Ad-TIMP3 decreased the migration (b) and invasion (c) ability of CT26 cells (compared with Ad-Null, *Po0.05). Representative micrographs of the transwell migration (d) and invasion (e) were shown ( 200 magnification).

Article Snippet: The TIMP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, Control, Virus, Migration

Journal: Cancer gene therapy

Article Title: Tissue inhibitor of metalloproteinases-3 transfer suppresses malignant behaviors of colorectal cancer cells.

doi: 10.1038/cgt.2012.70

Figure Lengend Snippet: Figure 4. Ad-TIMP3 infection decreased the tumor-formation ability of cancer cells. In the in invo tumor-formation assay, Ad-TIMP3 was used to treat the cancer cells at 25 MOI before the cells were implanted into the right flank of the mice. When the tumors can be touched, they were measured and recorded for the volumes. (a) The tumor growth curve. Ad-TIMP3-treated CT26 cells formed tumors were much smaller than that of the control group. (b) At the end of the observation, by tumor weight, Ad-TIMP3 pretreatment suppressed the tumor-formation ability of CT26 cells. (compared with Ad-Null, *Po0.05; #Po0.01). (c) Tumor growth curve during the Ad-TIMP3 treatment. Ad-TIMP3 significantly delayed the growth of tumors (Ad-TIMP3 compared with Ad-Null, *Po0.01, #Po0.05); (d) Compared with control Ad-Null, the tumor weight in Ad-TIMP3 was much lower than that in control groups (compared with Ad-con, *Po0.01, #Po0.05 ). (e) Metastatic lesions formed in liver. Eighteen mice were randomized into three groups and injected with variously treated CT26 cells (1 105 cells each). Seven days later, mice were killed and the liver metastatic lesions were counted on the sections (H&E staining). The Ad-TIMP3 group has much fewer lesions than other groups.

Article Snippet: The TIMP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, Tube Formation Assay, Control, Injection, Staining

Journal: Diagnostic Pathology

Article Title: Promoter methylation and expression of TIMP3 gene in gastric cancer

doi: 10.1186/1746-1596-8-110

Figure Lengend Snippet: Gastric carcinoma TIMP3 promoter methylation and protein expression

Article Snippet: TIMP3 antibody and SP kit were purchased from Boster Biological Engineering Co., Ltd. (Dalian).

Techniques: Methylation, Expressing

Journal: Diagnostic Pathology

Article Title: Promoter methylation and expression of TIMP3 gene in gastric cancer

doi: 10.1186/1746-1596-8-110

Figure Lengend Snippet: TIMP3 protein immunohistochemisty (SP 400×): a, normal gastric tissue; b, early gastric cancer; c, advanced gastric cancer; d, transfer of lymph node.

Article Snippet: TIMP3 antibody and SP kit were purchased from Boster Biological Engineering Co., Ltd. (Dalian).

Techniques:

Journal: Diagnostic Pathology

Article Title: Promoter methylation and expression of TIMP3 gene in gastric cancer

doi: 10.1186/1746-1596-8-110

Figure Lengend Snippet: Relationship between advanced gastric cancer pathology TIMP3 methylation and its protein expression level

Article Snippet: TIMP3 antibody and SP kit were purchased from Boster Biological Engineering Co., Ltd. (Dalian).

Techniques: Methylation, Expressing

Journal: Frontiers in pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: FIGURE 1 | Effects of dabigatran on pulmonary metastasis of 4T1 breast cancer cells injected intravenously into BALB/c mice. Mice were treated with vehicle (black symbols) or dabigatran etexilate (grey symbols) as described in Section 2.5, injected with 4T1 breast cancer cells, and euthanized 24 h, 2 days, and 7 days after injection. In (A) quantitative analysis of pulmonary metastasis in mice, based on CellTracker Red fluorescence in murine lungs isolated from animals 24 h and 2 days after injection of 4T1 cancer cells, is shown. The representative pictures of lung parenchyma illustrating metastatic count in untreated mice 24 h and 2 days after i.v. injection (100 x) are shown in (B,D), respectively; the representative images of dabigatran-treated mice 24 h and 2 days after injection are shown in (C,E), respectively. In (F), quantitative analysis of pulmonary metastasis based on haematoxylin and eosin (H&E) staining 7 days after i.v. injection is shown. The representative images of H&E- stained lung cross-sections (100×) of mice not treated and treated with dabigatran etexilate are shown in (G,I), respectively, while the results of Ilastik segmentation of (G,I) are shown in (H,J) (blue: lung parenchyma, green: pulmonary metastases), respectively. Statistical analysis in (A) was performed using a two-way ANOVA test followed by Bonferroni post hoc tests, while the results in (F) were analysed with two-sided unpaired T test. The results are presented as the median ± IQR. The symbol *** denotes statistical significance at p < .001.

Article Snippet: Seeding Into the Lungs in the Murine Model of Experimental Metastasis Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Injection, Isolation, Staining

Journal: Frontiers in pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: FIGURE 2 | Pulmonary fibrin deposition, lag time of thrombin generation in plasma, and innate immunity activation markers. Mice were treated with vehicle (black symbols) or dabigatran etexilate (grey symbols) as described in Section 2.5, injected with 4T1 breast cancer cells, and euthanized at 24 h, 2 days, and 7 days after injection. (A) Lung cross-sections were stained with biotin-conjugated IgG fraction of polyclonal goat antiserum to mouse fibrin antibody as described in Section 2. The representative images of fibrin deposition in the lungs of 4T1 breast cancer cell-injected mice are given in AI (untreated, 24 h post i.v.), AII (dabigatran-treated, 24 h post i.v.), AIII (untreated, 2 days post i.v.), AIV (dabigatran-treated, 2 days post i.v.), AV (untreated, 7 days post i.v.), AVI (dabigatran-treated, 7 days post i.v.) (200x). Scanned images were segmented using Ilastik software and counted in ImageJ to determine the number of pixels corresponding to fibrin signal; (B) the thrombin activity was determined by thrombin generation assay in murine plasma, and expressed as a lag time to cleavage of fluorogenic substrate by free thrombin; (C) concentrations of interferon γ (IFNγ) and (D) complement iC3b subunit were determined in lung homogenates by ELISA kits. Statistical analysis was performed with Kruskal-Wallis (A) and two-way ANOVA (B–D) tests followed by appropriate post hoc tests, and the results were presented as median ± IQR. The symbols *, **, and *** denote statistical significance at p < .05, p < .01, and p < 0.001, respectively.

Article Snippet: Seeding Into the Lungs in the Murine Model of Experimental Metastasis Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Clinical Proteomics, Activation Assay, Injection, Staining, Software, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: FIGURE 3 | Effects of dabigatran on lung permeability and inflammation markers in the lungs of BALB/c mice injected with 4T1 breast cancer cells. Mice were treated with vehicle (black symbols) or dabigatran etexilate (grey symbols) as described in Section 2.5. (A) depicts retention of Evans blue (EB) in the lung parenchyma, reflecting lung permeability. Control mice did not receive cancer cells and were euthanized at the end of the dabigatran pre-treatment to exclude the possibility that dabigatran affected pulmonary permeability of healthy animals before their inoculation with 4T1 cancer cells. After dabigatran/vehicle pre-treatment, the other mice were injected intravenously with 4T1 breast cancer cells or the vehicle and euthanized 24 h, 2 days, and 7 days after injection. (B–D) show the levels of Ang-2, E-selectin, and MMP-9, respectively. (E) shows representative Western blot images of Ang-2, E-selectin, and MMP-9 with densitometric data after their normalization to the total protein used as loading control based on the stain-free technique as described in Materials and Methods. For (A–D), statistical analysis was performed with two-way ANOVA and an appropriate post hoc test. The symbols *, **, and *** denote statistical significance at p < .05, p < .01, and p < .001, respectively, and the data are depicted as the median ± IQR.

Article Snippet: Seeding Into the Lungs in the Murine Model of Experimental Metastasis Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Permeability, Injection, Control, Western Blot, Staining

Journal: Frontiers in pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: FIGURE 4 | Effects of dabigatran on endothelium function based on selected biomarkers of endothelial dysfunction in BALB/c mice injected with 4T1 breast cancer cells. Mice were treated with vehicle (black symbols) or dabigatran etexilate (grey symbols) as described in Section 2.5, injected with 4T1 breast cancer cells, and euthanized at 24 h, 2 days, and 7 days after 4T1 breast cancer cell injection. The panel of selected biomarkers of endothelial dysfunction (Ang-1 (A), Ang-2 (B), sTie-2 (C), sFLT-1 (D), SDC-1 (E), sE-sel (F), sICAM-1 (G), vWF (H), PAI-1 (I), t-PA (J), sP-sel (K), and THBS-1 (L)) was measured in the plasma using the microLC/MS- MRM method as described in Section 2. The data are presented as the median ± IQR. Statistical analysis was performed with two-way ANOVA (A,C–H,J–L) or Kruskal- Wallis (B,I) tests followed by appropriate post hoc tests. The symbols *, **, and *** denote statistical significance at p < .05, p < .01, and p < .001, respectively.

Article Snippet: Seeding Into the Lungs in the Murine Model of Experimental Metastasis Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Injection, Clinical Proteomics

Journal: Frontiers in pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: FIGURE 5 | Effects of dabigatran on thrombin-induced platelet reactivity measured ex vivo. In (A,D–F), mice were treated with vehicle (black symbols) or dabigatran etexilate (grey symbols) as described in Section 2.5, injected with 4T1 breast cancer cells, and euthanized at 24 h, 2 days, and 7 days after injection, whereas in (B,C), washed blood samples of healthy mice were pre-incubated with dabigatran ex vivo. (A) shows the mean concentration of dabigatran in the animal plasma at the time of the euthanasia (approximately 6–8 h after the last oral dabigatran gavage). (B) shows the expression of an active form of GPIIb/IIIa on the surface of platelets in whole blood obtained from healthy animals. The blood samples isolated from these healthy animals were washed, pre-treated ex vivo with dabigatran at concentrations of 1, 10, 30, and 100 ng ml−1, and activated with bovine thrombin at a dose of .025 or .1 U ml−1. The data in (B) are presented as the mean and SEM of 2–3 independent experiments. Dabigatran alone at these concentrations did not affect the expression of the active form of GPIIb/IIIa (data not shown). (C) shows the representative flow cytometry results presented in (B). In (D) and (E), expression of an active form of GPIIb/IIIa on the platelet surface is shown as the percentage of the total (parent) platelet population and median fluorescence intensity (MFI) on the platelet surface, respectively, presented after activation of with .025 U ml−1 of bovine thrombin of washed blood samples obtained from untreated and dabigatran-treated 4T1 breast cancer cell-injected mice. In (F), the representative flow cytometry data of (D,E) are shown. The data were analysed with one-way ANOVA (A) or two-way ANOVA (D,E) followed by appropriate post hoc test, and the results are presented as median ± IQR. The symbols *, **, and *** denote statistical significance at p < .05, p < .01, and p < .001, respectively.

Article Snippet: Seeding Into the Lungs in the Murine Model of Experimental Metastasis Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Ex Vivo, Injection, Incubation, Concentration Assay, Clinical Proteomics, Expressing, Isolation, Cytometry, Activation Assay

Journal: Frontiers in pharmacology

Article Title: Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

doi: 10.3389/fphar.2022.834472

Figure Lengend Snippet: FIGURE 6 | Protection of human pulmonary endothelial barrier compromised by IL-1β stimulation afforded by releasates of washed human platelets and the reversal of this effect by dabigatran. Human lung microvascular endothelial cell (HLMVEC) monolayers were treated with releasates derived from non-stimulated PLT (R [PLTq]) (A) or PLT stimulated with .1 U ml−1 thrombin (R [PLTthr]) (B) in the presence of 10 ng ml−1 IL-1β or without IL-1β. The effect of releasates derived from thrombin- stimulated PLT on the barrier integrity formed by HLMVECs in the presence of 30 ng ml−1 of dabigatran (R [PLTthr + D]) was also tested without IL-1β (C) or in the presence of IL-1β (D). The data are shown as the mean of 3–4 independent experiments and were analysed using analysis of covariance (ANCOVA) in the stable phase (II). Thrombin alone added in Tyrode buffer (.1 U ml−1; the final concentration of .01 U ml−1) did not affect endothelial barrier integrity.

Article Snippet: Seeding Into the Lungs in the Murine Model of Experimental Metastasis Thirty mice were pre-treated with dabigatran etexilate (Biorbyt, cat. no orb180748) by oral gavage twice daily at a total dose of 100 mg per kg of body weight per day in .05% natrosol, and the same number of mice simultaneously received the vehicle (.05% natrosol) for 3 consecutive days.

Techniques: Derivative Assay, Concentration Assay

Journal:

Article Title: TIMP-3 inhibits the procollagen N-proteinase ADAMTS-2

doi: 10.1042/BJ20060630

Figure Lengend Snippet: (A) A constant amount of ADAMTS-2 (12 nM) was incubated with a constant amount of N-TIMP-3 (200 nM) and with increasing amounts of heparin, prior to incubation with 3H-labelled type II procollagen, followed by SDS/PAGE and autofluorography. The amount of heparin used is plotted against the ratio of procollagen II compared with the pCα1(II)-form (which retains the C-propeptide, while the N-propeptide has been removed). (B) A constant amount of ADAMTS-2 (12 nM) was incubated with increasing amounts of N-TIMP-3 (0–1000 nM) and 100 μg/ml heparin, prior to incubation with 3H-labelled type II procollagen, followed by SDS/PAGE and autofluorography. A Table of the percentage of procollagen II processed at each concentration of N-TIMP-3 is shown below the autofluorogram. Data were plotted and an apparent Ki of 160 nM was calculated using GraphPad Prism 4 software. The Ki range was 11.4 to 309.3 nM and the standard error was 60.9 nM (95% confidence intervals).

Article Snippet: Human TIMP-1, -2, -3 and -4 (with IC 50 values of ∼3, 2.5, 2.5 and 2 nm against MMP-2 respectively) and anti-TIMP-3 antibody were obtained from R&D Systems.

Techniques: Incubation, SDS Page, Concentration Assay, Software

Journal:

Article Title: TIMP-3 inhibits the procollagen N-proteinase ADAMTS-2

doi: 10.1042/BJ20060630

Figure Lengend Snippet: (A) An autofluorogram is shown of 3H-labelled type II procollagen incubated with ADAMTS-2 without (0×) or in the presence of TIMP-3/ADAMTS-2 (1:1, 2:1, 5:1, or 10:1, mol/mol; labelled 1×, 2×, 5× and 10× respectively). SM, procollagen II starting material. (B) Autofluorograms are shown of 3H-labelled type I (left-hand panel) or type III (right-hand panel) procollagen incubated without (−) or in the presence (+) of ADAMTS-2 and TIMP-3. TIMP-3/ADAMTS-2 (50:1, mol/mol) when incubated together. (C) Western blot of TIMP-3 and ADAMTS-2 incubated together at ratios of 5:0, 0:1, 1:1, 2:1 and 5:1 (mol/mol), followed by immunoprecipitation with antibody against TIMP-3 (α-TIMP-3), and then Western blot analyses with α-TIMP-3 antibody, or with antibody against the protein C epitope (α-protein C), for detection of ADAMTS-2. (D) An autofluorogram is shown of 3H-labelled type I procollagen incubated with BMP1 alone (0×) or in the presence of a 50-fold excess (50×) of TIMP-3. SM, procollagen I starting material.

Article Snippet: Human TIMP-1, -2, -3 and -4 (with IC 50 values of ∼3, 2.5, 2.5 and 2 nm against MMP-2 respectively) and anti-TIMP-3 antibody were obtained from R&D Systems.

Techniques: Incubation, Western Blot, Immunoprecipitation

Journal:

Article Title: TIMP-3 inhibits the procollagen N-proteinase ADAMTS-2

doi: 10.1042/BJ20060630

Figure Lengend Snippet: (A) A constant amount of ADAMTS-2 (12 nM) was incubated with increasing amounts of N-TIMP-3 (0–1000 nM), prior to incubation with 3H-labelled type II procollagen, followed by SDS/PAGE and autofluorography. A Table of the percentage procollagen II processed at each concentration of N-TIMP-3 is shown below the autofluorogram. Data were plotted and an apparent Ki of 618 nM was calculated using GraphPad Prism 4 software. The Ki range was 411–826 nM and the standard error was 80.7 nM (95.5 confidence intervals). (B) A constant amount of ADAMTS-2 (4 nM) was incubated with increasing amounts of N-TIMP-3 (0–500 nM), prior to incubation with 3H-labelled type II procollagen, followed by SDS/PAGE and autofluorography. A Table of the percentage of procollagen II processed at each concentration of N-TIMP-3 is shown below the autofluorogram. Data were plotted and an apparent Ki of 602 nM was calculated using GraphPad Prism 4 software. The Ki range was 396–809 nM and the standard error was 84.4 nM (95.5 confidence intervals).

Article Snippet: Human TIMP-1, -2, -3 and -4 (with IC 50 values of ∼3, 2.5, 2.5 and 2 nm against MMP-2 respectively) and anti-TIMP-3 antibody were obtained from R&D Systems.

Techniques: Incubation, SDS Page, Concentration Assay, Software

Journal:

Article Title: TIMP-3 inhibits the procollagen N-proteinase ADAMTS-2

doi: 10.1042/BJ20060630

Figure Lengend Snippet: N-TIMP-3 was added to the culture media of MEF cells, to a final concentration of 250 or 800 nM, in the presence of 0.01% dextran sulfate. Following SDS/PAGE and electrotransfer, pro-α1(I)-derived chains in cell layer samples were probed using an antibody directed against sequences in the pro-α1(I) C-telopeptide domain.

Article Snippet: Human TIMP-1, -2, -3 and -4 (with IC 50 values of ∼3, 2.5, 2.5 and 2 nm against MMP-2 respectively) and anti-TIMP-3 antibody were obtained from R&D Systems.

Techniques: Concentration Assay, SDS Page, Electrotransfer, Derivative Assay

Journal: The Journal of Immunology Author Choice

Article Title: The Metalloproteinase ADAMTS5 Is Expressed by Interstitial Inflammatory Cells in IgA Nephropathy and Is Proteolytically Active on the Kidney Matrix

doi: 10.4049/jimmunol.2000448

Figure Lengend Snippet: Identification of matrix proteins by urine proteomics leads to the discovery of ADAMTS5 in IgAN kidneys. ( A ) LC-MS/MS proteomics of healthy donor and IgAN patient urine identified multiple ECM proteins present in IgAN urine (see Ref. ). Matrix proteins were selected using gene ontology (BinGO) and are presented as a protein-protein interaction network. Yellow and red proteins are increased in IgAN urine, gray are unchanged, and blue are decreased in comparison with healthy samples. Proteomics analysis including all identified proteins is described in detail in . ( B ) The mRNA expression of ADAMTS5 and TIMP3 was examined in a published microarray dataset comparing 25 IgAN patients with six healthy donor kidney biopsies. Microarray gene expression was retrieved directly from National Center for Biotechnology Information–Gene Expression Omnibus (GSE35488; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35488 ). Microarray data are available in . ADAMTS5 and TIMP3 were significantly regulated in IgAN . Differences were examined using standard t test using data curated and processed by NCBI-GEO as described in GSE35488; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35488 . The p values are shown. ( C ) ADAMTS5 protein immunofluorescence was measured in 13 healthy (kidney transplant donors) versus 20 IgAN patients. IgAN donors were biopsy diagnosed. ADAMTS5 staining was quantified using ADAMTS5-stained particle counting corrected to biopsy tissue area on ImageJ and values were compared using two-tailed t test. ( D ) For the 20 IgAN patients, ADAMTS5 immunofluorescence was correlated to their matching urine protein concentration (proteinuria; mg/dl) and eGFR (estimated glomerular filtration rate; ml/min/1.73 m 2 ) values. The r and p values were computed using nonparametric Spearman test given the dissimilar nature of the correlated values. ( E ) ADAMTS5 staining was also compared in the same IgAN biopsies but biopsies were grouped according to their histological MEST-C score. We focused on the (T) score (percentage of biopsy area affected by tubular atrophy or interstitial fibrosis, whichever is greater) and compared less affected T0 biopsies ( n = 7) with more affected T1/T2 biopsies ( n = 13). There was a significant increase in ADAMTS5 staining in T1/T2 specimens ( t test). ( F ) ADAMTS5 biopsy staining was also measured separately in the tubulointerstitium and glomeruli and compared in healthy versus IgAN biopsies. ADAMTS5 staining in the IgAN interstitium ( IgAN Interst ) is significantly increased in comparison with staining in IgAN glomeruli ( IgAN Glom ), as well as in comparison with healthy interstitium and glomeruli, which are NS different to each other (ANOVA with Fisher least significant difference, multiple comparison test). ( G and H ) Examples of ADAMTS5 immunofluorescence in IgAN and healthy kidney biopsies costained with relevant proteins. In (G) ADAMTS5 (red) was costained with ADAMTS1 (green) and TIMP3 (blue). TIMP3 staining was below the threshold of confident detection in either IgAN or healthy biopsies. In (H) ADAMTS5 (red) was costained with TIMP1 (green). ADAMTS5 is close but does not appear to colocalise with TIMP1 in affected tubulointerstitial areas. tub denotes renal tubules and glom denotes glomeruli. Note that ADAMTS5 is increased in areas of tubulointerstitial inflammatory infiltration and remodelling. IgAN glomeruli also contain ADAMTS5 + cells. In healthy biopsies, there were sporadic ADAMTS5-stained (red) cells in the interstitium [(G) healthy] and glomeruli [(H) healthy]. White-dashed boxes denote areas shown in higher magnification in lower panels.

Article Snippet: Sections were treated using acid-based Ag retrieval and dewaxing solution (Abcam) at 98°C for 1 h. Sections were thoroughly washed, blocked in 10% normal donkey serum (Dako) for 1 h, and subsequently incubated with anti-human primary Abs against the following proteins: ADAMTS5 (raised in rabbit), CD64, and vimentin (mouse) from Abcam and TIMP3 (mouse), myeloperoxidase, TIMP1, lumican (LUM), ADAMTS1, versican (VCAN), collagen-4, and CD206 (all goat) from Bio-Techne.

Techniques: Liquid Chromatography with Mass Spectroscopy, Comparison, Expressing, Microarray, Immunofluorescence, Staining, Two Tailed Test, Protein Concentration, Filtration