trypsin 3  (R&D Systems)

Journal: Oncotarget

Article Title: Trypsinogen 4 boosts tumor endothelial cells migration through proteolysis of tissue factor pathway inhibitor-2

doi:

Figure Lengend Snippet: RNAof human endothelial cells from ovarian (HOC-EC) and kidney (HKC-EC) carcinomas, and from non-neoplastic tissues such as skin, adrenal gland (HA-EC), derma and lung (D- and L-HMVEC) was reverse-transcribed and PCR-amplified. For the primer sequences see . A: Endothelial cells do not express PRSS1 and PRSS2. Top panel shows the primers (Cf and Cr) common to PRSS1, PRSS2 and PRSS3 used to generate the amplicon, and the restriction sites discriminating the three transcripts. The uncut amplicon and the fragments produced by its digestion with PstI, XhoI, PvuII or SacI enzymes were separated by gel electrophoresis. The lower panels show the results for two representative EC populations, demonstrating that only PvuII can cut, hence indicating that only PRSS3 is expressed. B: Endothelial cells express PRSS3, specifically the trypsinogen 4 isoform. The 5′ terminus alignment of PRSS3, trypsinogen 4 and mesotrypsinogen, is shown with the common (Df, Dr and Er) and the specific (E1 and E2) primers for the different isoforms. The gel electrophoresis pictures show the PCR product of the expected length for PRSS3 (170 bp; top panel) and for trypsinogen 4 (345 bp; middle panel) and the lack of mesotrypsinogen amplification (191 bp; bottom panel) for a number of representative EC populations. (Positive control, HT29 colon cancer cells ; negative control, no template).

Article Snippet: The direct hydrolysis of TFPI-2 by PRSS3 was monitored in a time course experiment, incubating 500 nM recombinant human TFPI-2 (rhTFPI-2, R&D Systems) with 20 nM active recombinant human PRSS3 (rhPRSS3, R&D Systems) at 37°C in assay buffer (50mM Tris, 0.15M NaCl, 10mM CaCl2, 0.05% Brj-35, pH 7.5), following the manufacturer’s recommendations.

Techniques: Reverse Transcription, Amplification, Produced, Nucleic Acid Electrophoresis, Positive Control, Negative Control

Journal: Oncotarget

Article Title: Trypsinogen 4 boosts tumor endothelial cells migration through proteolysis of tissue factor pathway inhibitor-2

doi:

Figure Lengend Snippet: HOC-EC were transfected with siRNA specific for trypsinogen 4 (Tryp4 siRNA), TFPI-2 (TFPI-2 siRNA) or not-targeting (NT siRNA). Trypsinogen 4 and TFPI-2 mRNAs were quantified by RT-qPCR 24 hours later (expression by NT siRNA HOC-EC was arbitrarily assumed as reference). Three different HOC-EC populations were investigated (populations 23, 25 and 27); shown here are representative results. A: Trypsinogen 4-silenced tumor-EC have a much larger amount of TFPI-2 protein. Protein lysates were prepared 24 hours after transfection. Western blot analysis was done to assess TFPI-2 protein in the total lysate, in the cells and in the extracellular matrix (ECM). Tubulin was used as internal control. Migration was assessed by a wound healing assay. B: TFPI-2-silenced tumor-EC close the wound faster. The wound healing assay was done 24h after transfection and wound closure was monitored by Cell ® Imaging Station. TFPI-2 protein was quantified by Western blot analysis. C: Active trypsinogen 4 cleaves TFPI-2 in a cell-free system. Recombinanthuman TFPI-2 (500 nM) was incubated at 37°C with 20 nM of active recombinant human PRSS3 (rhPRSS3). The reaction was stopped at the timesindicated and the TFPI-2 protein was analysed by western blot. The arrows indicate the different TFPI-2 digestion products. Short and long membrane exposure time are shown to better illustrate how intact rhTFPI-2 fades (two bands, top panel) and the appearance of a number of lower molecular weight products (middle panel). The same amount of TFPI-2 was incubated at 37°C without trypsinogen 4 (bottom panel, long exposure). D: Active trypsinogen 4 displaces TFPI-2 from the extracellular matrix of tumor-EC. The extracellular matrix deposited by HOC-EC was incubated for 1h at 37°C with different concentrations of rhPRSS3 and TFPI-2 protein was analyzed by western blot.

Article Snippet: The direct hydrolysis of TFPI-2 by PRSS3 was monitored in a time course experiment, incubating 500 nM recombinant human TFPI-2 (rhTFPI-2, R&D Systems) with 20 nM active recombinant human PRSS3 (rhPRSS3, R&D Systems) at 37°C in assay buffer (50mM Tris, 0.15M NaCl, 10mM CaCl2, 0.05% Brj-35, pH 7.5), following the manufacturer’s recommendations.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Control, Migration, Wound Healing Assay, Imaging, Incubation, Recombinant, Membrane, Molecular Weight