Journal: Journal of Biological Chemistry

Article Title: Smac/DIABLO Selectively Reduces the Levels of c-IAP1 and c-IAP2 but Not That of XIAP and Livin in HeLa Cells

doi: 10.1074/jbc.m401253200

Figure Lengend Snippet: FIG. 6. Smac does not promote the auto-ubiquitination of XIAP in vitro. The purified XIAP (without tag) was incubated for 2 h at 30 °C with or without different concentrations of Smac or with Smac A in the reconstituted ubiquitination reaction system (Ub Mix) consisting of 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2 mM Mg-ATP, 20 M mammalian ubiquitin, 100 nM rabbit E1, and 400 nM UbcH6 (E2). The reactions were stopped by adding equal volumes of 2 SDS sample loading buffer, and the products were subjected to SDS-PAGE followed by immunoblotting with anti-XIAP antibody.

Article Snippet: This paper is available on line at http://www.jbc.org 16963 at SO U T H E R N IL L IN O IS U N IV on M arch 5, 2015 http://w w w .jbc.org/ D ow nloaded from of human c-IAP2, and residues 244–263 of human XIAP, and the monoclonal antibody against human ubiquitin were purchased from R & D Systems.

Techniques: Ubiquitin Proteomics, In Vitro, Purification, Incubation, SDS Page, Western Blot

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P < 0.001) destruction of the network created by the HUVECs following the treatment with LY3039478 (1 µM). The concomitant administration of MMP13 counteracts significantly ( P < 0.01) drug effectiveness.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P < 0.001 calculated with Student’s t test. b NOTCH1 gene and its pro-angiogenic targets are overexpressed in human intrahepatic cholangiocarcinoma (iCCA). Levels of NOTCH1, DLL4, VEGFA, and MMP13 mRNA were significantly more elevated in iCCA ( n = 42) than corresponding nontumorous surrounding livers (SL; n = 42), as detected by quantitative reverse-transcription PCR. Number target (NT) = 2 −ΔCt , wherein ΔCt value of each sample was calculated by subtracting the average Ct value of the gene of interest from the average Ct value of the β-actin gene. Mann–Whitney test: vs SL, P < 0.0001. c Expression of the NOTCH1 gene correlates with mRNA levels of putative target genes (HES1, DLL4, VEGFA, and MMP13) in a collection of human intrahepatic cholangiocarcinoma (CCA) samples ( n = 42). Linear regression analysis was used. d Representative expression patterns of CK19, NOTCH1, HES1, DDL4, and MMP13 in human intrahepatic cholangiocarcinoma (iCCA) as detected by immunohistochemistry. Upper panels: CCA case (CCA1) showing strong, concomitant immunoreactivity for NOTCH1, HES1, DDL4, and MMP13. Lower panels: CCA specimens (CCA2) exhibiting low levels of NOTCH1, HES1, DDL4, and MMP13. As expected, both iCCA display robust immunolabeling for CK19 (a biliary marker). Magnification: ×200; scale bar = 100 μm. H&E hematoxylin and eosin staining.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing, Reverse Transcription, MANN-WHITNEY, Immunohistochemistry, Immunolabeling, Marker, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Matrix Metalloproteinase 7 Mediates Epithelial–Mesenchymal Transition to Promote Liver Fibrosis Through E-cadherin/β-catenin Pathway in Biliary Atresia

doi: 10.3390/ijms27052209

Figure Lengend Snippet: Increased MMP7 in BA patients correlated with the progression of liver fibrosis. Serum and liver samples of patients with non-BA ( n = 39) disease and BA ( n = 66) were collected. ( A ) Serum MMP7 levels were determined by ELISA. ( B ) Relative MMP7 mRNA levels in livers of BA and non-BA patients. ( C ) Representative images of immunostaining for MMP7 and quantitative analysis of MMP7 staining in liver tissue. Magnification: left panel: ×100; right panel: ×400. ( D ) Representative Masson trichrome staining images of METAVIR fibrosis stages (F1–F4) in BA patients. Scale bar: 400 μm. ( E – G ) Serum MMP7 levels, relative MMP7 mRNA levels and the expression intensity of MMP7 in liver of BA patients with moderate (F0–F2) or advanced liver fibrosis (F3–F4). ( H ) Spearman’s rank correlation analysis of MMP7 expression level with fibrotic marker genes, including COL1A1 , ACTA2 , TGFB1 , and TIMP1 .

Article Snippet: The activation of recombinant human MMP7 protein (R&D Systems, Minneapolis, MN, USA) was achieved through incubation with 4-aminophenylmercuric acetate (APMA; Sigma, St. Louis, MO, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Immunostaining, Staining, Expressing, Marker

Journal: International Journal of Molecular Sciences

Article Title: Matrix Metalloproteinase 7 Mediates Epithelial–Mesenchymal Transition to Promote Liver Fibrosis Through E-cadherin/β-catenin Pathway in Biliary Atresia

doi: 10.3390/ijms27052209

Figure Lengend Snippet: MMP7 gene correlation analysis and pathway enrichment in BA. ( A ) Distribution of correlation coefficients between MMP7 expression and all other genes across two independent BA datasets ( GSE15235 and GSE46960 ). ( B ) GSEA results using gene lists ranked by correlation with MMP7.

Article Snippet: The activation of recombinant human MMP7 protein (R&D Systems, Minneapolis, MN, USA) was achieved through incubation with 4-aminophenylmercuric acetate (APMA; Sigma, St. Louis, MO, USA).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Matrix Metalloproteinase 7 Mediates Epithelial–Mesenchymal Transition to Promote Liver Fibrosis Through E-cadherin/β-catenin Pathway in Biliary Atresia

doi: 10.3390/ijms27052209

Figure Lengend Snippet: EMT scores were associated with MMP7, liver fibrosis and survival in BA patients. Liver samples of patients with non-BA ( n = 39) disease and BA ( n = 66) were collected. ( A ) Representative image of immunostaining for EMT-related protein expression (including E-cadherin, Vimentin, S100A4) in liver biopsies samples. Magnification: left panel : ×100; right panel : ×400. ( B ) Percentage of samples according to the intensity and extension of EMT-related protein immunostaining in cholangiocytes from two groups. ( C ) EMT scores of cholangiocytes in non-BA and BA patients. ( D ) EMT scores in two subgroups divided by MMP7 expression intensity in BA patients. ( E ) Correlation analysis of fibrosis stage EMT scores in BA patients. Spearman’s rank correlation.

Article Snippet: The activation of recombinant human MMP7 protein (R&D Systems, Minneapolis, MN, USA) was achieved through incubation with 4-aminophenylmercuric acetate (APMA; Sigma, St. Louis, MO, USA).

Techniques: Immunostaining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Matrix Metalloproteinase 7 Mediates Epithelial–Mesenchymal Transition to Promote Liver Fibrosis Through E-cadherin/β-catenin Pathway in Biliary Atresia

doi: 10.3390/ijms27052209

Figure Lengend Snippet: MMP7 promoted EMT in an E-cadherin/β-catenin pathway-dependent manner. ( A ) Protein expression of E-cadherin in HIBEpiCs after treatment with different concentrations of MMP7 for 24 h. ( B ) Level of sE-cadherin in the cell culture supernatant of HIBEpiCs after MMP7 treatment. ( C ) Western blot analysis of β-catenin protein in the nucleus or cytoplasm of HIBEpiCs. ( D ) Immunofluorescence staining shows the subcellular localization of β-catenin in HIBEpiCs. Scale bar:50 μm. ( E ) TOP/FOP-Flash luciferase activity of MMP7-treated cells. ( F ) Effect of the MMP7 and ICG-001 on β-catenin downstream target genes expression (including SNAI1 , SNAI2 , MMP7 ) in HIBEpiCs. ( G , H ) Effect of the MMP7 and ICG-001 on mRNA and protein expression of EMT-related markers (including E-cadherin, Vimentin, S100A4) in HIBEpiCs. ( I ) ELISA analysis of serum level of sE-cadherin in patients. ( J ) Spearman’s rank correlation analysis of serum sE-cadherin and serum MMP7 in BA patients. ( K ) Representative images of immunostaining of β-catenin in cholangiocytes of non-BA and BA patients. Magnification: left panel : ×100; right panel : ×400. ( C – H ) MMP7 concentration: 80 ng/mL; ICG-001 concentration: 25 μM. Data are presented as mean ± SD.

Article Snippet: The activation of recombinant human MMP7 protein (R&D Systems, Minneapolis, MN, USA) was achieved through incubation with 4-aminophenylmercuric acetate (APMA; Sigma, St. Louis, MO, USA).

Techniques: Expressing, Cell Culture, Western Blot, Immunofluorescence, Staining, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunostaining, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Matrix Metalloproteinase 7 Mediates Epithelial–Mesenchymal Transition to Promote Liver Fibrosis Through E-cadherin/β-catenin Pathway in Biliary Atresia

doi: 10.3390/ijms27052209

Figure Lengend Snippet: Fibrosis progression in chronic BA mice was accompanied by increased MMP7 expression and EMT. Liver and serum samples of the control group (i.p. saline) and chronic BA group (i.p. RRV+ anti-Ly6G) were collected on days 14, 21, and 42 postnatally ( n = 5~7 mice in each group). ( A ) Representative micrograph of Masson trichrome staining of the liver from control and chronic BA mice. Quantification of collagen staining in the liver of control and chronic BA mice. Magnification: ×40. ( B , C ) Serum level and hepatic mRNA expression of MMP7 in control and chronic BA mice. ( D ) Representative images of immunostaining for MMP7 expression in liver (Days 42). ( E ) Representative images of immunostaining for EMT-related protein expression (including E-cadherin, Vimentin, S100A4) in liver (Days 42). ( F ) Serum level of sE-cadherin in mice ( n = 5 mice in each group). ( G ) Representative images of immunostaining β-catenin in cholangiocytes of mice. Magnification: left panel : ×100; right panel : ×400. Data are presented as mean ± SD.

Article Snippet: The activation of recombinant human MMP7 protein (R&D Systems, Minneapolis, MN, USA) was achieved through incubation with 4-aminophenylmercuric acetate (APMA; Sigma, St. Louis, MO, USA).

Techniques: Expressing, Control, Saline, Staining, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Matrix Metalloproteinase 7 Mediates Epithelial–Mesenchymal Transition to Promote Liver Fibrosis Through E-cadherin/β-catenin Pathway in Biliary Atresia

doi: 10.3390/ijms27052209

Figure Lengend Snippet: MMP7 blockade attenuated liver fibrosis and EMT procession in chronic BA mice. ( A ) Schematic representation of chronic BA mice injected with anti-MMP7 antibody or IgG on days 21–25 and liver specimens collected on day 42 ( n = 5 mice in each group). ( B ) Representative micrographs of Masson trichrome stained liver tissue sections. Quantification of collagen staining in the liver. Magnification: ×40. ( C ) Representative images of immunostaining for EMT-related protein expression (including E-cadherin, Vimentin, S100A4) in the liver (Arrows indicate biliary epithelial cells). ( D ) Serum level of sE-cadherin in mice ( n = 5 mice in each group). ( E ) Representative images of immunostaining β-catenin in cholangiocytes of mice. ( F ) Serum level of MMP7 in mice ( n = 5 mice in each group). ( G ) Hepatic mRNA expression of MMP7 in mice ( n = 5 mice in each group). Magnification: left panel : ×100; right panel : ×400. Data are presented as mean ± SD.

Article Snippet: The activation of recombinant human MMP7 protein (R&D Systems, Minneapolis, MN, USA) was achieved through incubation with 4-aminophenylmercuric acetate (APMA; Sigma, St. Louis, MO, USA).

Techniques: Injection, Staining, Immunostaining, Expressing

Journal: Antibodies (Basel, Switzerland)

Article Title: Characterization of Active MMP9 in Chronic Inflammatory Diseases Using a Novel Anti-MMP9 Antibody.

doi: 10.3390/antib12010009

Figure Lengend Snippet: Figure 1. Activation of MMP9 by distinct proteases in vitro. Western blots for pro-MMP9 (92 kDa, red) and active MMP9 (84 kDa, green) after incubation with active (A) MMP7, (B) MMP12, (C) pepsin A, and (D) plasmin. Below each lane is the MMP9 activity towards gelatin expressed as gelatin fluorescence (arbitrary units) per minute. DQ, dye-quenched; MMP, matrix metalloproteinase.

Article Snippet: Recombinant active MMP7, MMP12, pepsin A, or plasmin (R&D Systems) was titrated against recombinant pro-MMP9 in MMP9 reaction buffer (150 mM NaCl, 10 mM Hepes pH 7.5, 0.05% brij-035, and 5 mM CaCl2).

Techniques: Activation Assay, In Vitro, Western Blot, Incubation, Activity Assay

Journal: Cancer Research

Article Title: LOXL2-Mediated Matrix Remodeling in Metastasis and Mammary Gland Involution

doi: 10.1158/0008-5472.can-10-2868

Figure Lengend Snippet: Figure 4. LOXL2 regulates TIMP1. A, Western blot analysis of LOXL2 protein expression and TIMP1 in concentrated CM from 4T1/MDA-MB-231 cells expressing control or LOXL2 shRNA constructs, or transfected with siLOXL2 (compared with mock-transfected control). B, immunofluorescent images of tumor sections from mice bearing 4T1/MDA-MB-231 control or shLOXL2 tumors stained with TIMP1 (green) and 40,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 100 mm. C, quantification of MDA-MB-231 control or siLOXL2 breast cancer cells þ/ recombinant human TIMP1 invading through matrigel towards a chemoattractant. *P < 0.05. D, invasive branching structures formed by MDA-MB-231control or siLOXL2 cells grown in 3D Matrigel, treated recombinant human TIMP1. Scale bar, top; 500 mm, bottom; 200 mm.

Article Snippet: The gels were transferred to polyvinylidene difluoride membranes (Millipore) and probed with antibodies specific to human LOXL2, TIMP1, b-actin (Abcam), mouse LOXL2 (Santa Cruz Biotechnology Inc.), or TIMP1 (R&D Systems).

Techniques: Western Blot, Expressing, Control, shRNA, Construct, Transfection, Staining, Recombinant

Journal: Cancer Research

Article Title: LOXL2-Mediated Matrix Remodeling in Metastasis and Mammary Gland Involution

doi: 10.1158/0008-5472.can-10-2868

Figure Lengend Snippet: Figure 5. High LOXL2 expression and activity during mammary gland involution. A, qRT-PCR analysis of LOXL2 mRNA expression levels in FVB mouse mammary gland samples at different stages of development (wV ¼ week virgin, dP ¼ day pregnancy, dL ¼ day lactation, dI ¼ day involution). n ¼ 3 mice per time point. * P < 0.05. B, Western blot analysis of LOXL2 and TIMP1 protein expression in lysates prepared from mice in (A). C, LOXL2 enzymatic activity in blood serum from mice in (A). n ¼ 3 mice per time point.

Article Snippet: The gels were transferred to polyvinylidene difluoride membranes (Millipore) and probed with antibodies specific to human LOXL2, TIMP1, b-actin (Abcam), mouse LOXL2 (Santa Cruz Biotechnology Inc.), or TIMP1 (R&D Systems).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot