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