Journal: Molecular carcinogenesis

Article Title: Knockdown of Notch1 inhibits nasopharyngeal carcinoma cell growth and metastasis via downregulation of CCL2, CXCL16, and uPA.

doi: 10.1002/mc.23082

Figure Lengend Snippet: FIGURE 5 Inhibition of Notch1 expression decreases the production of CCL2, CXCL16, uPA, and uPAR. A, The expression of CCL2 protein was significantly inhibited in Notch1 shRNA‐transfected cells. B, The expression of CXCL16 in CNE2 cells was decreased by more than half in Notch1 shRNA‐transfected cells compared with the control cells. In CNE1 cells, knockdown of Notch1 expression also led to a decrease of CXCL16, but the result was not significantly different. C, The expression of the uPAR protein was markedly inhibited in Notch1 shRNA‐transfected cells. D, Knockdown of Notch1 decreased uPA expression by about two‐fold. Columns, mean of triplicate assays; bars, SEM. * and ** represent P < .05, and P < .01, respectively, indicates a significant difference compared with the EV control. All the data are from at least three independent experiments. EV, empty vector; uPAR, urokinase plasminogen activator receptor

Article Snippet: Anti‐human monocyte chemoattractant protein 1 (MCP‐1/CCL2, #MAB279), C‐X‐C motif chemokine ligand 16 (CXCL16, #MAB976), urokinase plasminogen activator (uPA; #MAB1310), and uPA receptor (uPAR; #MAB807) monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN).

Techniques: Inhibition, Expressing, shRNA, Transfection, Control, Knockdown, Plasmid Preparation

Journal: Molecular carcinogenesis

Article Title: Knockdown of Notch1 inhibits nasopharyngeal carcinoma cell growth and metastasis via downregulation of CCL2, CXCL16, and uPA.

doi: 10.1002/mc.23082

Figure Lengend Snippet: FIGURE 6 uPA acts as a key mediator in Notch1‐induced NPC cell proliferation and invasion. A, The scatterplot shows that uPA gene was highly expressed in NPC tissues by Human Genome U133 Plus 2.0 Array.34 B, The knockdown efficiency of uPA in CNE1 cells, compared with EV control cells, was determined by qPCR. C, CNE1 cell proliferation was examined by the MTS assay. D, uPA‐shRNA‐transfected cells resulted in the significant upregulation of E‐cadherin protein expression and a reduction in vimentin expression. Cyclin D and cyclin E expression were markedly decreased in uPA knockdown cells. E, uPA‐shRNA‐transfected cells (left panel) resulted in the low penetration of cells through the Matrigel‐coated membrane, compared with control cells. Invasive Index (%) was calculated (right panel) according to the manufacturer's instructions. The data are from three independent experiments. Columns, mean of triplicate assays; bars, SEM. EV, empty vector; NPC, nasopharyngeal carcinoma; shRNA, short hairpin RNA; uPA, urokinase plasminogen activator. *P < .05, and **P < .01, respectively, indicates a significant difference compared with the EV control [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Anti‐human monocyte chemoattractant protein 1 (MCP‐1/CCL2, #MAB279), C‐X‐C motif chemokine ligand 16 (CXCL16, #MAB976), urokinase plasminogen activator (uPA; #MAB1310), and uPA receptor (uPAR; #MAB807) monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN).

Techniques: Knockdown, Control, MTS Assay, shRNA, Transfection, Expressing, Membrane, Plasmid Preparation

Journal: Neuro-Oncology

Article Title: Identification of genomic and molecular traits that present therapeutic vulnerability to HGF-targeted therapy in glioblastoma

doi: 10.1093/neuonc/noy105

Figure Lengend Snippet: YYB-101 treatment impedes tumor growth in vivo and downregulates important cellular molecular effectors. (A) Kaplan–Meier survival curves and representative boxplot of vehicle vs YYB-101-treated (5 mpk, 20 mpk) groups in BALB/c nude mice. P-values were calculated using the log-rank test for survival curve and Student’s t-test for boxplot. (B) Representative immunohistochemical images of p-MET, p-GAB1, p-FAK, MMP2, uPA/plasminogen, and Ki-67 stainings. Scale bars: p-MET, p-GAB1, p-FAK, MMP2, and uPA/plasminogen, 200 μm; Ki-67, 100 μm.

Article Snippet: Tissue sections of paraffin-embedded specimens were stained with the following primary antibodies: p-MET (Y1234/1235) (E9P0077, Enogene), p-GAB1 (Y307) (3234p, Cell Signaling), p‒focal adhesion kinase (FAK) (Y397) (ab4803, Abcam), MMP2 (6E3F8) (ab86607, Abcam), and urokinase-type plasminogen activator (uPA)/urokinase (AF1310, R&D system).

Techniques: In Vivo, Immunohistochemical staining

Journal: Molecular cancer

Article Title: MicroRNA-940 suppresses prostate cancer migration and invasion by regulating MIEN1.

doi: 10.1186/1476-4598-13-250

Figure Lengend Snippet: Figure 5 miR-940 targets MIEN1 and affects MMP-9, uPA and VEGF expression in a cellular context-dependent manner. (A-B) Expression of the downstream targets of MIEN1 upon transfection of Pre-miR-NT or Pre-miR-940 in DU-145 (A) and Anti-miR-NT or Anti-miR-940 in PWR-1E (B) at both the translational and transcriptional levels as shown by western blotting (A,i, B,i) and qPCR (A,ii, B,ii) respectively. (C-D) qPCR showing the expression of MIEN1 and the effectors upon transfection with Anti-miR-940 in PC-3 (C) and with Pre-miR-940 in PC-3 (D).***P ≤0.001; **P ≤0.01; *P ≤0.05.

Article Snippet: The following antibodies and reagents were used: Mouse monoclonal and mouse polyclonal MIEN1 (Abnova; antibody specificity tested and proven in previous studies[15,17]), rabbit polyclonal MIEN1 (Life Technologies; antibody specificity tested in previous studies[15]), mouse monoclonal GAPDH (Santa Cruz Biotechnology), rabbit monoclonal pNF-κB p65 S536 and rabbit polyclonal MMP-9 (Cell Signaling Technology), mouse monoclonal VEGF and uPA (R&D Systems), mouse monoclonal Alexa Fluor 594 conjugated Phalloidin (Life Technologies), mouse monoclonal E-cadherin (BD Biosciences), Vimentin (supernatant developed in mouse and tested against human antigen, Developmental Studies Hybridoma Bank), anti-mouse and anti-rabbit IgG (Promega), AlexaFluor 488 goat anti-mouse IgG and AlexaFluor 594 goat anti-mouse IgG (Life Technologies) sheep anti-DIG-AP antibody and NBT-BCIP ready-to-use tablets (Roche), sheep serum (Jackson ImmunoResearch), rabbit IgG, BSA, levamisole hydrochloride, Tris-HCl (pH 7.4), nuclease free water, SSC buffer, Xylene, Tween-20, Nuclear Fast Red, Hematoxylin and Eosin (Sigma-Aldrich) and Permount and PBS (Thermo Fisher Scientific).

Techniques: Expressing, Transfection, Western Blot