Journal: PloS one

Article Title: Apoptosis induced by knockdown of uPAR and MMP-9 is mediated by inactivation of EGFR/STAT3 signaling in medulloblastoma.

doi: 10.1371/journal.pone.0044798

Figure Lengend Snippet: Figure 1. Transfection with pU, pM and pUM in combination with radiation specifically down regulates expression of uPAR and MMP-9 and induces apoptosis. Daoy and D283 cell lines were transfected with either transfection reagent alone (control, Con), pSV (scrambled vector) or gene specific shRNA’s as described in materials and methods. A) RT-PCR analysis and western blotting was carried to determine the expression levels of uPAR and MMP-9 in cell transfected with reagent, pSV, pU-, pM- and pUM-transfected Daoy and D283 cells (with and without radiation, 8 Gy). The experiments were repeated three times and representative images were shown. The immunoblots were stripped and re-probed with GAPDH as a loading control. Semi-quantitative RT-PCR analysis was carried out to detect mRNA levels of uPAR and MMP-9 using specific primers. B) Protein band and PCR amplicon intensities were quantified by densitometry analysis using ImageJ software (National Institutes of Health). The levels of uPAR and MMP-9 protein were normalized to GAPDH levels in mock-transfected cells. Columns: mean of triplicate experiments; bars: s.d.; *p,0.01, significant difference from pSV-transfected cells. C) 72 hrs after Transfection (with or without radiation), the cells were trypsinized and analyzed by flow cytometry to measure the number of apoptotic TUNEL-positive cells using the APO-BrdU TUNEL Assay kit. The percent apoptotic cells from each treatment are represented and the mean 6 s.d. from three separate experiments was represented; *p,0.05 and ** p,0.01 were

Article Snippet: Plasmid expressing full length uPAR gene was purchased from Origene (Rockville, MD), and recombinant MMP-9 was purchased from R&D systems (R&D Systems, Minneapolis, MN).

Techniques: Transfection, Expressing, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Amplification, Software, Flow Cytometry, TUNEL Assay

Journal: PloS one

Article Title: Apoptosis induced by knockdown of uPAR and MMP-9 is mediated by inactivation of EGFR/STAT3 signaling in medulloblastoma.

doi: 10.1371/journal.pone.0044798

Figure Lengend Snippet: Figure 2. uPAR and MMP-9 gene silencing induced mitochondrial apoptosis critically depends on Bak activation and cytochrome C release into cytosol in Daoy and D283 cells. A) 72 hrs after transfecting Daoy and D283 cells with pU, pM and pUM (with or without radiation, 8 Gy), the cells were collected and analyzed by western blotting with the indicated antibodies. The membrane was reprobed with GAPDH to confirm equal protein loading. B) Sub-cellular fractions were prepared from the transfected cells as described in materials and methods. Cytochrome C release from mitochondria into the cytosol and mitochondrial Bak levels were determined by Western blotting of mitochondrial and cytosolic protein extracts. Membranes were re-probed with a-tubulin (cytosolic marker) or COX IV (mitochondrial marker). C) Mitochondrial membrane potential damage was evaluated using MitoLight green (Millipore). To determine the initiation of mitochondrial apoptosis in shRNA transfected Daoy and D283 cells, we carried out FACS analysis of mitochondrial membrane potential by sorting cells as described in materials and methods. Graphs represents percent number of cells showing reduced mitochondrial membrane potential in Daoy and D283 cell transfected with shuPAR, shMMP9 and shuPAR- MMP9 (treated with and without radiation). Each experiment is repeated 3 times * p,0.05 were statistically significant compared to pSV-transfected cells. A total of 10,000 cells were sorted per treatment. doi:10.1371/journal.pone.0044798.g002

Article Snippet: Plasmid expressing full length uPAR gene was purchased from Origene (Rockville, MD), and recombinant MMP-9 was purchased from R&D systems (R&D Systems, Minneapolis, MN).

Techniques: Activation Assay, Western Blot, Membrane, Transfection, Marker, shRNA

Journal: PloS one

Article Title: Apoptosis induced by knockdown of uPAR and MMP-9 is mediated by inactivation of EGFR/STAT3 signaling in medulloblastoma.

doi: 10.1371/journal.pone.0044798

Figure Lengend Snippet: Figure 3. Transcriptional silencing of uPAR and MMP-9 activates caspase-3, caspase-9 and PARP. Activity of caspase-3 and caspase-9 in Daoy and D283 cells was determined using A) caspase-3 and B) caspase-9 colorimetric activity kits. Total cell lysates collected from transfected cells (with and with radiation) were collected, incubated with the respective peptide substrate, and treated with caspase conjugate p-nitroaniline (Ac- DEVD-pNA). Further, activity was measured at 405 nm using a microplate reader. Y-axis shows the percent activity of the respective caspase by normalizing the control to 100%. Bars represent the means 6 s.d. from three independent experiments. * p,0.01 and ** p,0.05 were statistically significant compared to control and pSV-transfected cells. C) Immunoblot analysis of proteins isolated from cells treated with pU, pM and pUM (with and without radiation) was carried out to determine the activities of caspase-3, caspase-9 and PARP cleavage. D) The above immunoblots were stripped and re-probed with survivin, XIAP and cIAP1 specific antibodies, to determine the expression levels of inhibitory proteins. doi:10.1371/journal.pone.0044798.g003

Article Snippet: Plasmid expressing full length uPAR gene was purchased from Origene (Rockville, MD), and recombinant MMP-9 was purchased from R&D systems (R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Transfection, Incubation, Control, Western Blot, Isolation, Expressing

Journal: PloS one

Article Title: Apoptosis induced by knockdown of uPAR and MMP-9 is mediated by inactivation of EGFR/STAT3 signaling in medulloblastoma.

doi: 10.1371/journal.pone.0044798

Figure Lengend Snippet: Figure 4. Silencing uPAR and MMP-9 inhibits nuclear levels and activation of STAT3 and NF-kB p65 (Rel-A). A) Total cell lysates were evaluated by immunoblotting to determine the expression of NFkB p65 (Rel-A), IkBa, total and phosphorylated forms of EGFR and STAT3. Uniform loading of the respective protein was confirmed by re-probing the membrane with b-actin antibody. B) Nuclear levels of phosphorylated STAT3 and Rel-A were determined by analyzing the nuclear extracts isolated from transfected cells by western blotting. C–D) Nuclear extracts were prepared from cells transfected with pU, pM and pUM (with and with radiation), and the DNA binding activity of the nuclear extracts to C) STAT3 and D) NFkB p65 probe was determined using Electrophorotic Mobility gel Shift Assay. All experiments were repeated three times. doi:10.1371/journal.pone.0044798.g004

Article Snippet: Plasmid expressing full length uPAR gene was purchased from Origene (Rockville, MD), and recombinant MMP-9 was purchased from R&D systems (R&D Systems, Minneapolis, MN).

Techniques: Activation Assay, Western Blot, Expressing, Membrane, Isolation, Transfection, Binding Assay, Activity Assay, Gel Shift

Journal: PloS one

Article Title: Apoptosis induced by knockdown of uPAR and MMP-9 is mediated by inactivation of EGFR/STAT3 signaling in medulloblastoma.

doi: 10.1371/journal.pone.0044798

Figure Lengend Snippet: Figure 5. uPAR and MMP-9 activates EGFR and STAT3; Inhibition of STAT3 and NF-kB 65 induces apoptosis in Daoy and D283 cells. To determine possible cross-talk between STAT3 and Rel-A during apoptosis, each gene was downregulated using specific siRNA followed by determination of the nuclear levels of STAT3 and Rel-A in both Daoy and D283 cells. A) Medulloblastoma cells transfected with full-length uPAR expressing plasmid or/and incubated with recombinant MMP-9 (25 gg/ml), were either blocked with EGFR neutralizing antibody or non-specific isotype IgG to determine the role of the extracellular proteases in activation of EGFR and STAT3. Western blot analysis was carried out on cell lysates isolated from Daoy and D283 to determine the levels of phosphorylated EGFR and STAT3. B) Western blot analysis was carried out on the nuclear extracts isolated from STAT3-downregulated cells. Membranes were probed with phosphorylated forms of STAT3 and Rel-A. C) Similarly, nuclear extracts isolated from Rel-A downregulated cells were evaluated by Western blotting to detect phosphorylated forms of Rel-A and STAT3 in the nucleus. D) Chromatin immunoprecipitation assay was carried out with the nuclear extracts isolated from Day and D283 cells transfected with either STAT3 siRNA or pUM plasmid. Chromatin was immunopreciptated with STAT3 and analyzed by PCR using primers specific for Bcl-2 promoter region to determine the STAT3 recruitment at Bcl-2 promoter sequences. Chromatin immunoprecipitated with isotype IgG was used as negative control. Input DNA was confirmed by amplifying the Bcl-2 promoter form the chromatin aliquot collected prior to immunoprecipitation step. doi:10.1371/journal.pone.0044798.g005

Article Snippet: Plasmid expressing full length uPAR gene was purchased from Origene (Rockville, MD), and recombinant MMP-9 was purchased from R&D systems (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Incubation, Recombinant, Activation Assay, Western Blot, Isolation, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control

Journal: PloS one

Article Title: Apoptosis induced by knockdown of uPAR and MMP-9 is mediated by inactivation of EGFR/STAT3 signaling in medulloblastoma.

doi: 10.1371/journal.pone.0044798

Figure Lengend Snippet: Figure 6. Inhibition of uPAR and MMP-9 induces apoptosis in vivo. Daoy cells were implanted intracranially in nude mice, which were treated with control pSV or pUM (150 mg) either alone or in combination with radiation as described in Materials and Methods. A–B) Paraffin-embedded brain tumor sections from mice that received control shRNA or pUM either alone and combination of radiation were analyzed by immunohistochmeical analysis using antibodies for A) uPAR and B) MMP-9 followed by treatment with secondary antibody conjugated with

Article Snippet: Plasmid expressing full length uPAR gene was purchased from Origene (Rockville, MD), and recombinant MMP-9 was purchased from R&D systems (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, In Vivo, Control, shRNA

Journal: Journal of the American Society of Nephrology

Article Title: Sphingomyelinase-Like Phosphodiesterase 3b Expression Levels Determine Podocyte Injury Phenotypes in Glomerular Disease

doi: 10.1681/asn.2013111213

Figure Lengend Snippet: Figure 5. Interaction of suPAR and SMPDL3b. (A) Co-IP experiments performed in HEK293 cells showing an interaction between GFP- SMPDL3b and FLAG-uPAR (F-uPAR; upper panel) but not between GFP-SMPDL3b and FLAG-b3 integrin (F-b3; lower panel). FLAG- empty vector (F-C) was used as a negative control (upper panel). (B) Endogenous immunoprecipitation showing interaction of SMPDL3b and suPAR in glomeruli isolated from mice injected with PBS or LPS. E1, eluate from PBS-injected mice; E2, eluate from LPS-injected mice; I1, input (glomerular lysate) from PBS-injected mice; I2, input (glomerular lysate) from LPS-injected mice; IP, immunoprecipitation; WB, Western blot. (C) Competitive Co-IP experiments performed in HEK293 show that increasing amounts of GFP-SMPDL3b interfere with the interaction of FLAG-uPAR/suPAR and GFP-b3 integrin. However, transfection of GFP-empty vector used as a control does not affect the interaction between uPAR/suPAR and b3 integrin. GFP, green fluorescent protein.

Article Snippet: The biopsy tissue specimens were manually microdissected,41–43 and glomerular gene expression profiling was performed as previously described.15 Measurements of suPAR Circulating suPAR levels were determined in the sera from 53 patients with FSGS from the FSGS clinical trial25 and 30 patients with type 1 diabetes and normoalbuminuria, 34 patients with type 1 diabetes and microalbuminuria, and 10 patients with type 1 diabetes and macroalbuminuria from the FinnDiane study cohort using Quantikine Human uPAR Immunoassay (R&D Sys- tems) performed following the manufacturer’s protocol.

Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation, Negative Control, Immunoprecipitation, Isolation, Injection, Western Blot, Transfection, Control

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