Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: Cleavage profile of the HMPV F: A fluorogenic peptide mimicking the cleavage site of HMPV F was incubated with the indicated proteases and cleavage was monitored by the increase of fluorescence at 390 nm. RFU = relative fluorescence units. StDev = Standard Deviation.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Incubation, Fluorescence

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: IC50 values of all protease/peptide combinations.: Fluorogenic peptides mimicking the cleavage sites of A/CA/04/09 H1N1, A/Japan/305/1957 H2N2 HA, A/Aichi/2/68 H3N2 HA, A/Vietnam/1203/2004 H5N1 LPAI HA, A/Vietnam/1204/2004 H5N1 HPAI HA, A/Taiwan/2/2013 H6N1 HA, A/Shanghai/2/2013 H7N9 HA, A/Hong Kong/2108/2003 H9N2 HA and HMPV F were incubated with the indicated proteases and different SPINT2 concentrations. Cleavage was monitored by the increase of fluorescence at 390 nm and the resulting Vmax values were used to calculate the IC50 values as described in the “Material and Methods” section. (A) IC50 values of influenza A fluorogenic cleavage site peptide mimics. Concentrations are in nanomolar. (B) IC50 values of the HMPV F cleavage site peptide mimic. Concentrations are in picomolar. NT = Not tested.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Incubation, Fluorescence

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: SPINT2 inhibits cleavage of HA protein expressed in 293T cells. Cells were transfected with plasmids encoding for the indicated HA and allowed to express the protein for ~18 h. The recombinant proteases were incubated for 15 min with the indicated SPINT2 concentrations and subsequently added to the cells for 10 min (trypsin) or 90 min (matriptase and KLK5). Western blots were performed and the HA 1 band was quantified using ImageJ. (A) Quantification of the HA1 band comparing the signal intensity of the 0 nM SPINT2 samples against 10 nM and 500 nM SPINT2 of the respective HA/protease combination. Three independent experiments were carried out and the western blots of each experiment were analyzed. Quantifications were conducted as described in the methods section. (B – D) Western blots showing the cleavage of (B) A/CA/04/09 H1N1 HA by matriptase and KLK5 at different SPINT2 concentration, (C) A/Aichi/2/68 H3N2 HA by KLK5 at different SPINT2 concentration and (D) A/Shanghai/2/2013 H7N9 HA by matriptase and KLK5 at different SPINT2 concentrations. Statistical analysis was performed using a non-paired student's t-test comparing the samples tested with 10 nM SPINT2 against the respective sample incubated with 500 nM SPINT2. Error bars indicate standard deviation. * indicates p = < 0.05.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Transfection, Recombinant, Incubation, Western Blot, Concentration Assay, Standard Deviation

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: TMPRSS2, HAT, matriptase and KLK5 cleave HMPV F and SPINT2 is able to prevent cleavage by exogenous proteases . HMPV F was either expressed alone or co-transfected with protease and allowed to express for ~ 18 h. Cells were then metabolically starved of cysteine and methionine followed by radioactive S35 labeling of protein for 4 h in the presence of TPCK-trypsin or specified protease. SPINT2 treated proteases were incubated at room temperature for 10 min and placed onto cells for 4 h. Radioactive gels were quantified using ImageQuant software with percent cleavage equal to [ ( F 1 F 0 + F 1 ) x 100 ] . (A and B) Co-transfected proteases TMPRSS2, HAT and matriptase are able to cleave HMPV F (n = 4) while C and D) exogenous proteases KLK5 and matriptase but not KLK12 are able to cleave HMPV F (n = 5). (E and F) SPINT2 prevented cleavage of HMPV F by trypsin, KLK5 and matriptase at nm concentrations demonstrated by the loss of the F 1 cleavage product (n = 3). Statistical analysis was performed using a one-way ANOVA followed by a student's t-test with a bonferroni multiple comparisons test correction. P < 0.05 *, P < 0.005 **, P < 0.0005 ***, P < 0.0001 ****. N values represent independent replicates for each treatment group. Error bars represent SD.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Transfection, Metabolic Labelling, Labeling, Incubation, Software

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: SPINT2 inhibits HA-mediated cell-cell fusion. VERO cells were transfected with plasmids encoding for (A) A/CA/04/09 H1N1 HA or (B) A/Shanghai/2/2013 H7N9 HA and allowed to express the protein for ~18 h. Recombinant matriptase and KLK5 were incubated with different SPINT2 concentrations for 15 min and then added to the HA-expressing cells for 3 h. After 3 h the cells were briefly treated with cell fusion buffer at pH5, washed, supplemented with growth medium and returned to the incubator for 1 h to allow fusion. HA protein was detected using HA-specific primary antibodies and a secondary fluorogenic Alexa488 antibody. Nuclei were stained using DAPI. Magnification 40x. (C) VERO cells expressed A/Vietnam/1204/2004 H5N1 HA that was cleaved during its maturation process in the cell. SPINT2 was added at 0 nM or 500 nM at the time of transfection. Magnification 25x.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Transfection, Recombinant, Incubation, Expressing, Staining

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: SPINT2 reduces IAV growth in cell culture. MDCK cells were transfected with plasmids encoding for human matriptase or human TMPRSS2 and allowed to express the proteins for ~18 h. Cells expressing human matriptase (B) or human TMPRSS2 (C) were then infected with A/CA/04/09 H1N1 at a MOI of 0.1 and different SPINT2 concentration were added to each well. Non-transfected cells to which trypsin was added served as a control (A). (D) MDCK cells were infected with A/X31 H3N2 at an MOI of 0.1 and trypsin was added to assist viral propagation. Different SPINT2 concentration were added as indicated. After 48 h of infection the supernatants were collected and used for an immuno-plaque assay to determine the viral loads. Experiment was repeated three times and each dot represents the viral titer of a single experiment. Statistical analysis was performed using a non-paired student's t-test comparing the control (0 h) against the respective sample. Error bars indicate standard deviation. * indicates p = < 0.05. Extended horizontal line within the error bars represents mean value of the three independent replicates.

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Cell Culture, Transfection, Expressing, Infection, Concentration Assay, Control, Plaque Assay, Standard Deviation

Journal: Virology

Article Title: SPINT2 inhibits proteases involved in activation of both influenza viruses and metapneumoviruses

doi: 10.1016/j.virol.2020.01.004

Figure Lengend Snippet: Viral titers measured in the infection studies: Table shows the average viral titers and standard deviation calculated from the 3 independent biological replicates depicted in Fig. 6 , Fig. 7 . (A) Titers and standard deviation from infection studies shown in Fig. 5 . (B) Titers and standard deviation shown in Fig. 7 .

Article Snippet: The plasmids encoding for A/Shanghai/2/2013 (H7N9) HA, human TMPRSS2 and human matriptase were purchased from Sino Biological Inc.

Techniques: Infection, Standard Deviation

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: Specificity and potency of anti-PAR2 monoclonal antibody MEDI0618 . ( A ) Live staining of PAR2-expressing (1321N1-hPAR2.cl8) or non-expressing (1321N1 parental) cell lines with MEDI0618 directly conjugated to Alexa Fluor 647. Scale bar = 20 µm. ( B ) Flow cytometry of hPAR2 overexpressing cells (1321N1-hPAR2.cl8) or non-expressing cells (1321N1 parental cell line) or A549 cells endogenously expressing hPAR2 live labelled with MEDI0618. ( C and D ) Calcium imaging from 1321N1-hPAR2 cells pretreated with MEDI0618 hIgG or an isotype control protein. ( C ) Exemplar raw calcium trace from a single well pretreated with MEDI0618 or isotype control antibody both at 1 nM, followed by PAR2 agonist stimulation with matriptase (10 nM). ( D ) Antibody titration of the anti-PAR2 antibodies MEDI0618 or PAR650097 or an isotype control antibody. Data show the matriptase (10 nM)-mediated calcium signal after preincubation with antibody and normalized to the matriptase response in the absence of antibody treatment. ( E ) Calcium imaging from A549 cells pretreated with MEDI0618, isotype control protein or PAR1 inhibitors (ATAP2 + WEDE15 mAbs) followed by 10 nM thrombin (PAR1 agonist) stimulation. Data are presented as mean ± standard error of the mean, n = 4. The concentration of the inhibitor is shown on the x -axis. Data are normalized to the peak thrombin calcium response in the absence of inhibitor pretreatment.

Article Snippet: Recombinant human matriptase (R&D Systems) was diluted in 50 mM Tris-HCl plus 10% glycerol at a concentration of 23 μM prior to final dilution in assay buffer.

Techniques: Staining, Expressing, Flow Cytometry, Imaging, Control, Titration, Concentration Assay

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: PAR2 functional expression in human and mouse cells relevant to migraine . Whole well calcium imaging from primary human dural fibroblasts (HDuF), human dural microvascular endothelial (HDuMEC) and mouse brain endothelial (bEnd.3) cells. ( A , D and G ) PAR2 agonists concentration response curve in ( A ) HDuF, ( D ) HDuMEC and ( G ) bEnd.3 cells. ( B , E and F ) Effect of MEDI0618 and isotype control protein (IgG) on inhibition of matriptase-induced calcium signalling at 30 nM in ( B ) HDuF, ( E ) HDuMEC and ( H ) bEnd.3 cells. ( C , F and I ) Representative calcium imaging traces of 30 nM matriptase-evoked activity following MEDI0618 or isotype control protein preincubation in ( C ) HDuF, ( F ) HDuMEC and ( I ) bEnd.3 cells. ( J and K ) Single-cell calcium imaging from mouse trigeminal neuron cultures. ( J ) Pseudocolour images of fura-2 ratio intensity show a subset of trigeminal neurons activated by treatment with 10 µM LIGRLO in comparison to 20 mM KCl treatment. Scale bar = 20 µm. ( K ) Representation of fura-2 traces recorded from two individual neurons during acute LIGRLO (10 µM) or KCl (20 mM) treatment.

Article Snippet: Recombinant human matriptase (R&D Systems) was diluted in 50 mM Tris-HCl plus 10% glycerol at a concentration of 23 μM prior to final dilution in assay buffer.

Techniques: Functional Assay, Expressing, Imaging, Concentration Assay, Control, Inhibition, Activity Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: Matriptase protein and constructs. A, schematic drawing of full-length matriptase with the N-terminal transmembrane region (black), the SEA domain (green), two CUB domains (orange), four LDLRa domains (blue), and the C-terminal serine protease domain (purple). The cleaved activation loop rearranges to create the catalytically active protease. B, schematic drawing of the zymogen-locked form of matriptase characterized in this paper, consisting of only the serine protease domain with position R614A mutated to avoid activation (zSPD). C, a Coomassie-stained SDS-PAGE (10%) shows that zSPD (consisting of only the serine protease domain, as shown in B) is pure and the expected size (25–30 kDa). D, the sequence of the crystallized form of zSPD (zSPD-S805A) with an additional mutation in the active site, shown with matriptase numbering (sides) and chymotrypsin numbering (top). The mutations are color-coded: R614A (blue), N772Q (green), and S805A (blue).

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques: Construct, Activation Assay, Staining, SDS Page, Sequencing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: The zymogenicity factor of matriptase All values represent an average of at least three independent measurements.

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: The monoclonal antibodies aZ-mAb-6 and aZ-mAb-7 are not substrates for matriptase. Antibodies aZ-mAb-6 and aZ-mAb-7 were incubated with or without acSPD at a 1:1 molar ratio for 1 h at 37 °C. No cleavage of the antibodies is seen after incubation with matriptase.

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques: Bioprocessing, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: The monoclonal antibodies aZ-mAb-6 and -7 specifically inhibit human matriptase. The ability of aZ-mAb-4, -6, and -7 to inhibit human matriptase and other closely related serine proteases was tested in an activity assay. For all assays, an excess of antibody (400 nm) was preincubated with the proteases for 1 h at 37 °C before the addition of substrate. Human matriptase, acSPD (100 pm), was almost completely inhibited by aZ-mAb-6 (green line) and aZ-mAb-7 (black line), but not by aZ-mAb-4 (red line). The activity of mouse matriptase SPD (100 pm), human uPA (1 nm), hepsin (1 nm), and HGFA (5 nm) were not significantly inhibited by any of the monoclonal antibodies. The graphs shown are representative of three replicates.

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques: Bioprocessing, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: A, the monoclonal antibodies aZ-mAb-6 and -7 inhibit matriptase activity in the protein extracts of transiently transfected cells. HEK293 cells were transiently transfected with empty expression vector (mock) or expression vectors encoding WT full-length matriptase together with WT HAI-2 or mutant HAI-2 C47F, respectively. Protein extracts were preincubated for 1 h at room temperature with 500 ng of antibody (anti-uPA, aZ-mAb-6, aZ-mAb-7, or no antibody), and a peptidolytic activity assay was subsequently carried out with 300 μm chromogenic substrate S-2288. The absorbance of the reaction mixture was measured at 405 nm continuously every 5 min for 5 h, and the mean velocity of the substrate reaction (in milli-absorbance units/min) was calculated. The figure shown is representative of three replicates. B, aZ-mAb-6 and -7 inhibit matriptase-mediated pro-HGF cleavage. Pro-HGF (60 nm final concentration) was incubated for 4.5 h at 37 °C with either 200 nm zSPD or 1 nm acSPD that had been preincubated with aZ-mAb-6, aZ-mAb-7, anti-uPA, or biotin-RQRR-CMK, as indicated above the gels. The processing of pro-HGF was visualized on Western blotting using an antibody that recognizes pro-HGF as well as its α and β chains. Antibody-only samples were prepared in parallel to show the cross-reactivity of the antibodies themselves with the secondary antibody.

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques: Bioprocessing, Activity Assay, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Concentration Assay, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors

doi: 10.1074/jbc.RA118.004126

Figure Lengend Snippet: The structure of zymogen matriptase, zSPD-S805A. A, the solved structure of zSPD-S805A (wheat) with the 60 loop, the 70 loop, the 170 loop, and the activation loop specified together with the residues in the catalytic triad and the S1 pocket (PDB entry 5LYO). B, the intact activation loop for zSPD-S805A (wheat) and the cleaved activation loop for activated matriptase (red). C, the zymogen triad for zSPD-S805A and activated matriptase, showing rearrangement of Asp804 (Asp194). D, alignment of zSPD-S805A (wheat) and activated matriptase (gray), where the loops undergoing conformational changes are marked in red for activated matriptase. The S1 pocket is not formed in the zymogen.

Article Snippet: The proteases and their respective chromogenic substrates in HBS-PEG buffer were as follows: acSPD (100 p m ) and mouse matriptase catalytic domain (100 p m ; R&D Systems) with S-2288 (500 μ m ); full-length human uPA (1 n m ; ProSpec) with S-2444 (500 μ m ); full-length human HGFA (5 n m ; Sino Biological Inc.) with Spectrozyme FVIIa (500 μ m ), where HGFA was activated by thrombin (1:100 molar ratio) at 37 °C for 1 h until thrombin was blocked with 10 μg/ml Dextran Sulfate; and human hepsin (1 n m ) with S-2366 (500 μ m ) and in HBS-BSA buffer.

Techniques: Activation Assay

Journal: Oncology Reports

Article Title: Downregulation of matriptase suppresses the PAR-2/PLCγ2/PKC-mediated invasion and migration abilities of MCF-7 breast cancer cells

doi: 10.3892/or.2021.8198

Figure Lengend Snippet: Effect of matriptase on the TPA-mediated expression of MMP-9 in MCF-7 cells. (A) MCF-7 cells were transfected with control and matriptase siRNAs for 24 and 48 h, and matriptase expression was confirmed by western blot analysis. (B) Transfected MCF-7 cells were treated with TPA for 24 h, and the levels of MMP-9 protein expression were detected by western blotting. (C) Total RNA was isolated from MCF-7 cells and MMP-9 mRNA levels were detected using reverse transcription-quantitative PCR. Data represent the mean ± SEM of three independent experiments. # P<0.01 vs. untreated control; *P<0.01 vs. TPA. TPA, 12- O -tetradecanoylphorbol-13-acetate; siRNA, small interfering RNA.

Article Snippet: Matriptase-specific antibodies were obtained from R&D Systems (cat. no. MAB3946).

Techniques: Expressing, Transfection, Control, Western Blot, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Oncology Reports

Article Title: Downregulation of matriptase suppresses the PAR-2/PLCγ2/PKC-mediated invasion and migration abilities of MCF-7 breast cancer cells

doi: 10.3892/or.2021.8198

Figure Lengend Snippet: Effect of BAPTA-AM on TPA-induced PKC activation and MMP-9 expression, and the effect of matriptase on PLCγ2 phosphorylation, in MCF-7 cells. MCF-7 cells were treated with BAPTA-AM (20 µM) and TPA for (A) 40 min (to evaluate PKC activation) or (B) 24 h (to analyze MMP-9 expression). PKC isozyme and MMP-9 protein levels were detected by western blotting in MCF-7 cells. (C) MCF-7 cells were transfected with control and matriptase siRNAs for 24 h, after which PLCγ2 activation/levels of p-PLCγ2 were detected using western blotting. Data represent the mean ± SEM of three independent experiments. # P<0.01 vs. control siRNA-treated. TPA, 12- O -tetradecanoylphorbol-13-acetate; PKC, protein kinase C; siRNA, small interfering RNA; PLC, phospholipase C; p-, phosphorylated; Na K ATPase, sodium/potassium ATPase.

Article Snippet: Matriptase-specific antibodies were obtained from R&D Systems (cat. no. MAB3946).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Western Blot, Transfection, Control, Small Interfering RNA

Journal: Oncology Reports

Article Title: Downregulation of matriptase suppresses the PAR-2/PLCγ2/PKC-mediated invasion and migration abilities of MCF-7 breast cancer cells

doi: 10.3892/or.2021.8198

Figure Lengend Snippet: Effect of matriptase on TPA-induced PKC activation and MAPK signaling in MCF-7 cells. (A) MCF-7 cells were transfected with control and matriptase siRNAs for 24 h, followed by incubation with 20 nM TPA for 40 min. PKC isozyme levels in cell membrane fractions were analyzed by western blotting. (B) Transfected cells were treated with 20 nM TPA for 30 min and cell lysates were prepared for western blotting to evaluate the MAPK signaling pathway. (C) Additionally, transfected cells were treated with TPA, and after 4 h of incubation, cytoplasmic lysates were prepared; expression of upstream signaling molecules NF-κB, p-IκBα, IκBα, p-IKKαβ, IKKα and IKKβ were than analyzed via western blotting. TPA, 12- O -tetradecanoylphorbol-13-acetate; PKC, protein kinase C; siRNA, small interfering RNA; p-, phosphorylated; Na K ATPase, sodium/potassium ATPase.

Article Snippet: Matriptase-specific antibodies were obtained from R&D Systems (cat. no. MAB3946).

Techniques: Activation Assay, Transfection, Control, Incubation, Membrane, Western Blot, Expressing, Small Interfering RNA

Journal: Oncology Reports

Article Title: Downregulation of matriptase suppresses the PAR-2/PLCγ2/PKC-mediated invasion and migration abilities of MCF-7 breast cancer cells

doi: 10.3892/or.2021.8198

Figure Lengend Snippet: Effect of matriptase on TPA-induced NF-κB and AP-1 activation in MCF-7 cells. (A) MCF-7 cells were transfected with control and matriptase siRNAs for 24 h, followed by incubation with 20 nM TPA. After 4 h of incubation, western blot analysis was performed to determine the nuclear levels of p50 and AP-1 (p-c-Jun) subunits. (B) NF-κB-Luc or (C) AP-1-Luc reporter and the Renilla luciferase thymidine kinase reporter vector were co-transfected into MCF-7 cells. Cells transfected with matriptase siRNA for 24 h with TPA and promoter activity of NF-κB and AP-1 was measured with dual-luciferase reporter assays. Data represent the mean ± SEM of three independent experiments. # P<0.01 vs. untreated control; *P<0.01 vs. TPA. TPA, 12- O -tetradecanoylphorbol-13-acetate; siRNA, small interfering RNA; p-, phosphorylated; PCNA, proliferating cell nuclear antigen; AP-1, activator protein-1.

Article Snippet: Matriptase-specific antibodies were obtained from R&D Systems (cat. no. MAB3946).

Techniques: Activation Assay, Transfection, Control, Incubation, Western Blot, Luciferase, Plasmid Preparation, Activity Assay, Small Interfering RNA

Journal: Oncology Reports

Article Title: Downregulation of matriptase suppresses the PAR-2/PLCγ2/PKC-mediated invasion and migration abilities of MCF-7 breast cancer cells

doi: 10.3892/or.2021.8198

Figure Lengend Snippet: Effect of matriptase on the TPA-mediated invasiveness and migration of MCF-7 cells. (A) MCF-7 cells (3×10 5 ) were transfected with control and matriptase siRNAs, and seeded into the upper chamber of a Matrigel-coated Transwell insert; TPA was added to the lower chamber. (B) Transfected cell lysates were subjected to migration analyses. At 24 h post-incubation, invasive and migratory cells were stained and counted; magnification, ×10. Data represent the mean ± SEM of three independent experiments. # P<0.05 vs. untreated control; *P<0.05 vs. control + TPA. TPA, 12- O -tetradecanoylphorbol-13-acetate; siRNA, small interfering RNA.

Article Snippet: Matriptase-specific antibodies were obtained from R&D Systems (cat. no. MAB3946).

Techniques: Migration, Transfection, Control, Incubation, Staining, Small Interfering RNA

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 1. Expression analysis of matriptase, HAI-1, HAI-2, and prostasin in B cancer cells by reverse- transcription/qPCR (a,b), western blotting (c–e), and flow cytometry (f). (a) Bar graph of relative mRNA expression levels of matriptase (Mat), HAI-1, HAI-2, prostasin (Pro) in Daudi (n = 4), Namalwa (n = 5), Ramos (n = 3), Raji (n = 3), JeKo-1 (n = 3), and RS4;11 cells (n = 2) using GAPDH as the reference. The prostasin bars do not appear in the bar graph, as the actual qPCR readouts were registered as “N/A” by the instrument. (b) Bar graph of mRNA quantity ratio of HAI-2 to matriptase after normalization with the GAPDH level in each cell line in (a). (c) Western blotting images of matriptase (Ab: A300-221A), HAI-2, and GAPDH. Twenty micrograms of total protein from the cell lysate of each individual culture (including 2 repeats) were analyzed. Daudi, lanes 1–3; Namalwa, lanes 4–6; Ramos, lanes 7–9. Top panel, matriptase (Mat); middle panel, HAI-2; bottom panel, GAPDH. (d) Densitometry bar graph of relative protein quantities of matriptase and HAI-2 using GAPDH as the reference. (e) The quantitative ratio of HAI-2 to matriptase in each cell line. (f) Flow cytometry histogram of matriptase expression evaluation in Ramos cells. The Ramos cells (4 × 105) were labeled with the matriptase antibody as described in the Materials and Methods section. The matriptase-positive cells are shown in the PE-A subset (blue peak). Cells without the matriptase antibody labeling (red peak) were not detected in the PE-A subset and were used as the gating control.

Article Snippet: Rabbit anti-human prostasin sera or pre-immune rabbit sera [7] and the matriptase antibody (A6135, ABclonal Technology, Woburn, MA, USA) were used as the primary antibodies at 1:100 dilution.

Techniques: Expressing, Reverse Transcription, Western Blot, Cytometry, Flow Cytometry, Labeling, Antibody Labeling, Control

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 2. Prostasin exosomes reduce matriptase quantity in B cancer cells. (a) Western blot images of matriptase (Ab: A300-221A) in samples from the cell lysate (top panel) and the conditioned media (bottom panel) after incubation with prostasin exosomes (Pro) or exosomes without prostasin (KO). The Daudi cells (2 × 105 cells each) were incubated with the exosomes in 50 µL of OPTI-MEM I/2%FBS (lanes 1–4) or RPMI medium (lanes 5–8) overnight. One-half of each cell lysate or 40 µL of each media supernatant were analyzed. (b) Western blot images of GAPDH from (a). (c) Densitometry of relative intensities of matriptase in the cell lysate or media (d). Data presented are the average intensity of lanes 1, 3, 5, 7 versus that of lanes 2, 4, 6, 8 after normalization with GAPDH in (b). (e) Western blot images of matriptase (top panel; Ab: sc-365482) in the Daudi, Namalwa, and Ramos cells treated with exosomes isolated from the HEK293T cells. Cells (2.5 × 105) were co-cultured with prostasin exosomes (Pexo, lanes 3, 6, 9) or vector exosomes (Vexo, lanes 2, 5, 8) in 100 µL of OPTI-MEM I/2%FBS. Cells without exosomes (None, lanes 1, 4, 7) were cultured in the same conditions. Bottom, GAPDH western blot image. (f) Bar graph of (e) expressed as the relative intensities of matriptase

Article Snippet: Rabbit anti-human prostasin sera or pre-immune rabbit sera [7] and the matriptase antibody (A6135, ABclonal Technology, Woburn, MA, USA) were used as the primary antibodies at 1:100 dilution.

Techniques: Western Blot, Incubation, Isolation, Cell Culture, Plasmid Preparation

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 3. B cell matriptase quantity reduction by wild-type prostasin. (a) Western blot images of matriptase (Ab: sc-365482) and GAPDH in the Daudi (top two panels), Ramos (middle two panels), and Namalwa (bottom two panels) cells treated with exosomes isolated from the Calu-3 cells and sublines with over-expressed prostasin or variants. Calu-3, parent cells; KO, subline with prostasin

Article Snippet: Rabbit anti-human prostasin sera or pre-immune rabbit sera [7] and the matriptase antibody (A6135, ABclonal Technology, Woburn, MA, USA) were used as the primary antibodies at 1:100 dilution.

Techniques: Western Blot, Isolation

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 5. Ectopic expression of prostasin in B cancer cells. (a) Western blot analysis of transient expression of prostasin (P) or vector alone (V) in the Daudi, Namalwa, and Ramos cells. The lysate from 2 × 105 cells of each type was analyzed. Top panel, matriptase (Ab: sc-365482); middle panel, prostasin; bottom panel, GAPDH. (b) Flow cytometry analysis of Namalwa sublines with tetracycline-induced prostasin expression or vector alone. Red peak (vector-alone cells) and sky-blue peak (prostasin-expressing cells) are samples without the prostasin antibody incubation. Orange peak (vector-alone cells) and green peak (prostasin-expressing cells) are samples incubated with the prostasin antibody. All samples were incubated with a secondary antibody conjugated with the fluorophore Cy3, and 10,000 cells of each sample were analyzed in a CytoFLEX S flow cytometer. The data were analyzed with FlowJo™software v10.8.1 and are presented in the histogram. (c) Western blot analysis of NamalwaTR sublines. One hundred thousand cells of each sample were analyzed. Lanes 1 and 4 or V, samples of the vector control subline; lanes 2 and 5 or P, samples of the subline with the wild-type prostasin; lanes 3 and 6 or M, samples of the subline with a serine active-site mutant prostasin. Left panel, cells were grown in OPTI-MEM I/2%FBS with 1 µg/mL tetracycline (with tet); right panel, cells were grown without tetracycline (no tet) for 8 days. Top two panels, matriptase antibody (sc-365482); bottom two panels, prostasin antibody. (d) Western blot analysis of tet-conditioned media from (c). Two hundred milliliters of the conditioned media were precipitated with trichloroacetic acid (TCA) (final 16.7%) at 4 ◦C overnight. The pellet was collected via centrifu- gation and analyzed. The membrane was blotted with the AF3946 human matriptase/ST14 catalytic domain antibody.

Article Snippet: Rabbit anti-human prostasin sera or pre-immune rabbit sera [7] and the matriptase antibody (A6135, ABclonal Technology, Woburn, MA, USA) were used as the primary antibodies at 1:100 dilution.

Techniques: Expressing, Western Blot, Plasmid Preparation, Flow Cytometry, Incubation, Cytometry, Software, Control, Mutagenesis, Membrane

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 6. Impact of prostasin–matriptase cascade activation on B cancer cells. (a) Bar graph of cell count for two consecutive days of B cells treated with exosomes. Namalwa, n = 7; Ramos, n = 6; Raji, n = 5; Jeko-1, n = 6. * denotes p < 0.05. (b) Growth curves of NamalwaTR-Vec and NamalwaTR-Pro cells under tetracycline induction. Left graph, cells were set at 2.5 × 105/mL on day 0 and cultured in the growth medium containing 10%FBS for 4 days. Right graph, on day 4 (reset, indicated by the arrow), the cells were diluted in OPTI-MEM I/2%FBS to 5 × 105/mL and cultured for another 5 days. Tetracycline at 1 µg/mL was added into the culture on day 0 and maintained through culturing. n = 4 for each cell line, and * denotes p < 0.05. (c) Trypsin-like serine protease activity in the conditioned media of NamalwaTR-Vec and NamalwaTR-Pro cells (n = 4). Data were analyzed in Excel with student’s t test. * denotes p < 0.05 between the two sample groups. (d) Bar graph of annexin-V-positive cells analyzed by flow cytometry. Cells under tetracycline induction were cultured for various times (week 1, n = 3; week 2, n = 4; week 3, n = 3) and subjected to direct labeling of annexin V conjugated with fluorophore allophycocyanin (APC). Ten thousand cells for each sample were analyzed on the CytoFLEX S flow cytometer. Propidium iodide staining and FSC/SSC discrimination were used for gating the live singlets, which were further analyzed for annexin V staining. (e) Bar graph of migrated cells treated with exosomes for 24 h. * denotes p < 0.05. (f) Bar graph of migrated Namalwa sublines with the induction of prostasin expression for 2–4 days and reconditioned in RPMI medium for 1 day before seeding in Transwells for migration (n = 7). * denotes p < 0.05. (g) Bar graph of invaded cells treated with exosomes for 24 h. (h) Bar graph of invaded Namalwa sublines (n = 5) treated as in (f). * denotes p < 0.05. (i) Gelatin zymography and western blot analysis. Top panel, the Ramos cells (2 × 106) in 500 µL of RPMI/0.1%BSA were treated with vector exosomes (Vexo), prostasin exosomes (Pexo), or a purified recombinant human matriptase serine protease domain (r-Mat SPD) overnight. One-fifth of the cell lysate (lanes 1–3) or 20 µL of the conditioned medium (lanes 4–6) were analyzed. The Vexo or Pexo exosomes or the r-Mat SPD alone were incubated in RPMI/0.1%BSA and used as controls (lanes 7–9). The clear bands at ~70 kDa marked by a filled arrow are matriptase. These were recognized by matriptase antibodies (middle panel). Unidentified bands with gelatinase activity marked at * locations in lanes 4, 5, 7, 8 are inherited from the exosomes, as shown in the samples with the exosomes alone (lanes 7 and 8). The band marked by the white circle is unknown. Bands at ~28 kDa marked by an unfilled arrow are r-Mat SPD. Bottom panel is GAPDH, which is detected only in the cell lysate, not in media samples or the controls without cells. (j) Gelatin zymography of B cancer cells treated as described in (i). The matriptase gelatinase activity is decreased in the cell lysate (lanes 2, 6, 10, 14) but increased in the corresponding media samples (lanes 4, 8, 12, 16) upon Pexo treatment in comparison to that of the Vexo-treated samples (in lysate, lanes 1, 5, 9, 13; in media, lanes 3, 7, 11, 15), correspondingly.

Article Snippet: Rabbit anti-human prostasin sera or pre-immune rabbit sera [7] and the matriptase antibody (A6135, ABclonal Technology, Woburn, MA, USA) were used as the primary antibodies at 1:100 dilution.

Techniques: Activation Assay, Cell Counting, Cell Culture, Activity Assay, Cytometry, Labeling, Staining, Expressing, Migration, Zymography, Western Blot, Plasmid Preparation, Recombinant, Incubation, Comparison

Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: A. Western blot of normal immortalized human oral keratinocytes (NOK, lane 1) and eight human SCC cell lines (HN6-HN31, lanes 2–9). Positions of molecular weight markers (kDa) at left and matriptase (Mat) and GAPDH loading control at right. Expression of ST14 , encoding matriptase (B), and SPINT1 , encoding the matriptase inhibitor, hepatocyte growth factor inhibitor (HAI)-1 (B') in eight gene expression array studies of human SCC of the head and neck, or skin. Data are expressed as fold change relative to corresponding normal tissue. *, P<0.05. **, P<0.01. Matriptase (C and D) and Ki67 (C' and D') immunohistochemistry of skin (C and C') and oral (D and D') SCC demonstrating matriptase expression in proliferating tumor cells at sites of invasion (examples with arrowheads). E. Expression of HGFR , encoding c-Met, in the eight array studies analyzed in B and B'. *, P<0.05; **, P<0.01. F. Anatomical location of the 72 SCC biopsies used for analyzing matriptase and c-Met expression. Numbers in the pie chart indicate number of tumors analyzed from each location. Representative immunohistochemistry for matriptase (G–J) and c-Met (G'–J') in serial sections from four arrayed HNSCCs showing co-expression of matriptase and c-Met at the invasive front (examples with arrowheads) and other locations. Stars show examples of stroma. Size bars: C–D' 50 μm, G–J' 200 μm.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Western Blot, Molecular Weight, Control, Expressing, Gene Expression, Immunohistochemistry

Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: Matriptase (A–D) and Ki67 (A'–D') immunohistochemistry of normal epidermis (A and A'), hyperplasia (B and B'), dysplasia (C and C'), and squamous cell carcinoma (D and D') during murine chemical multi-stage carcinogenesis. Expression of matriptase in proliferating basal keratinocytes of hyperplastic and dysplastic lesions (examples with arrowheads in B and C) as well as in tumor cells at the invasive front (examples with arrowheads in D), but not in basal keratinocytes of normal epidermis (examples with arrowheads in A). The keratinocyte and tumor cell compartments expressing matriptase have high rates of proliferating cells as shown by expression of Ki67 (examples with arrowheads B', C', and D'). Stars in A–D' indicates the location of the dermis or the tumor stroma. E–E". Double immunofluorescence staining of squamous cell carcinoma for matriptase (E, red, examples with arrowheads) and c-Met (E', green, examples with arrowheads). Overlay in E" shows co-localization of matriptase and c-Met on the tumor cell surface (yellow, examples with arrowheads). Stars in E–E" shows location of the tumor stroma. Size bars: A–B', C, and D, 50 μm; C', D', E–E", 25 μm.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Immunohistochemistry, Expressing, Double Immunofluorescence Staining

Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: A. Efficient activation of recombinant proHGF/SF by the activated matriptase serine protease domain in solution. proHGF/SF (40 nM) was incubated with 4 (lane 2), 2 (lane 3), 1 (lane 4), and 0.5 (lane 5) nM matriptase or vehicle (lanes 1 and 6) for 1 h at 37 °C. Lanes 1 and 7 are single-chain proHGF/SF and two-chain HGF standards, respectively. Positions of single-chain proHGF/SF, heavy (hcHGF) and light (lcHGF) chains of two-chain HGF/SF are indicated right. Molecular weight markers (kDa) are indicated left. B. Elevated matriptase expression in cultured primary K5-matriptase transgenic keratinocytes. Cell lysates (lanes 1 and 2) and conditioned medium (lanes 3 and 4) from newborn wildtype (lanes 1 and 3) and littermate K5-matriptase +/0 (lanes 2 and 4) keratinocyte cultures and matriptase expression was analyzed by Western blot. Positions of full-length (Mat L) and SEA domain-processed (Mat S) forms of matriptase are indicated with arrowheads. Positions of molecular weight markers (kDa) are indicated at left. C. Elevated matriptase expression increases c-Met and Gab1 phosphorylation in response to single-chain proHGF/SF, but not to active two-chain HGF/SF. Primary keratinocytes from newborn wildtype (panels 1, 3 and 5 from top) and littermate K5-matriptase +/0 (panels 2, 4, and 6 from top) were treated with either 2.5 nM proHGF/SF (lanes 1–7) or active HGF/SF (lanes 8 and 9) for 0 (lane 1), 5 (lanes 8 and 9), 10 (lane 2), 20 (lane 3), 30 (lanes 4 and 7), 45 (lane 5), and 60 (lane 6) min in the absence (lanes 1–6, and 8) or presence (lanes 7 and 9) of the serine protease inhibitor, aprotinin. Phosphorylated c-Met (panels 1 and 2 from top), phosphorylated Gab1 (panels 3 and 4 from top), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (bottom two panels) were detected by Western blotting. D–J. Elevated matriptase expression amplifies the migratory response of primary keratinocytes to single-chain proHGF/SF, but not to active two-chain HGF/SF. Scrape wounds were generated in confluent monolayers of primary keratinocytes from newborn wildtype (WT) (D–F') and littermate K5-matriptase +/0 (K5) (G–I') mice, and the monolayers were treated with vehicle (D, D', G, and G'), 2.5 nM proHGF/SF (E, E', H, and H') or proHGF/SF with 2 μM aprotinin (Ap) (F, F', I, and I') for 24 h. Dashed yellow lines indicate the margins of denuded area at 0 h. Examples of keratinocytes that have migrated into the denuded area after 24 h are shown in E' and H'. Size bars: 100 μm. J. Quantitation of average migration distance of wildtype (blue bars) and littermate K5-matriptase +/0 keratinocytes (green bars) in response to vehicle, proHGF/SF, proHGF/SF and aprotinin (Ap), active HGF/SF and active HGF/SF with aprotinin. Results are shown as mean migration distance ± standard deviation of the mean.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Activation Assay, Recombinant, Incubation, Molecular Weight, Expressing, Cell Culture, Transgenic Assay, Western Blot, Phospho-proteomics, Protease Inhibitor, Generated, Quantitation Assay, Migration, Standard Deviation

Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: A–C'. Matriptase-induced follicular metaplasia depends on keratinocyte c-Met. Outwards appearance of untreated four (A–C) and 12 (A'–C') months-old control (A and A'), c-Met-sufficient K5-Mat (K5Mat;cMet+) (B and B'), and c-Met-deficient K5-Mat (K5Mat;cMet-) (C an C') mice. D–G. Loss of keratinocyte c-Met blocks matriptase-induced epidermal hyperplasia, dysplasia, and expression of stress keratin. Hematoxylin and eosin (D), BrdU (E), and keratin-6 (K6) (F) immunohistochemistry of epidermis from control (top panels), keratinocyte c-Met-deficient (second panels from top), c-Met sufficient K5-Mat (third panels from top), and c-Met-deficient K5-Mat mice (bottom panels) at 12 months of age. Arrowheads in D and F show location of the basal layer of the epidermis and stars in D and F show location of dermis. Arrowheads in E are examples of BrdU incorporating keratinocytes. G. Enumeration of BrdU incorporation in basal keratinocytes in the four groups of mice. P values by Student's t-test, two tailed. H–K. Matriptase-induced dermal fibrosis and hypercellularity is prevented by loss of keratinocyte c-Met. Masson's trichrome staining of skin of control (H), c-Met-deficient (I), c-Met sufficient K5-Mat (J), and c-Met-deficient K5-Mat mice (K) at 12 months of age. Size bars: 50 μm.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Control, Expressing, Immunohistochemistry, BrdU Incorporation Assay, Two Tailed Test, Staining

Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: A–D. Immunohistochemistry for phospho-S6 ribosomal protein (pS6) during DMBA and matriptase-induced multi-stage squamous cell carcinogenesis. mTor-dependent phosphorylation of S6 protein is confined to suprabasal keratinocytes in normal and hyperplastic epidermis, whereas S6 protein is phosphorylated in all keratinocyte layers of the dysplastic epidermis, and all tumor cells of squamous cell carcinomas. E and F. Representative examples of the outward appearance of K5-Mat mice treated with vehicle (E) or rapamycin (F) for 33 weeks after DMBA treatment. G and H. Histological appearance of H&E stained epidermis of vehicle-(top panel, G) or rapamycin-treated (middle panel, G) control mice, and of rapamycin-treated (bottom panel, G) and vehicle-treated (H) K5Mat mice. High magnification (I and J) of epidermis of vehicle- (I) and rapamycin- (J) treated mice. Examples of basal keratinocytes and tumor cells at the invasive front are indicated with arrowheads in G–J. Stars in I and J indicate tumor stroma and dermis, respectively. Size bars: A–G, 50 μm; H, 100 μm; I–J, 25 μm. K. Kaplan-Meier analysis of DMBA-induced epidermal tumor formation in rapamycin-treated K5-Mat (black lines, N=15), rapamycin-treated wildtype (green lines, N=15), vehicle-treated wildtype (blue lines, N=15), and vehicle-treated K5-Mat (red lines, N=15) littermate mice. P values were determined by the log-rank test, two-tailed. L. Enumeration of BrdU incorporation in basal keratinocytes of K5-Mat and wildtype littermate mice treated with vehicle or rapamycin. P values were determined by Student's t-test, two tailed.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Immunohistochemistry, Phospho-proteomics, Staining, Control, Two Tailed Test, BrdU Incorporation Assay

Journal: Oncogene

Article Title: c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase

doi: 10.1038/onc.2010.586

Figure Lengend Snippet: 1. Mesenchymal cells located in close proximity to c-Met- and matriptase-expressing basal keratinocytes with high tumorigenic potential secrete single-chain proHGF/SF into the pericellular microenvironment. 2. ProHGF/SF binds c-Met with high affinity on the keratinocyte cell surface. 3. Matriptase cleaves and converts single-chain proHGF/SF to signaling-competent two-chain HGF/SF. 4. Matriptase-cleaved two-chain HGF/SF undergoes a conformational change that enables c-Met activation by autophosphorylation. 5. Activation of c-Met leads to recruitment of Gab1 and other effectors of c-Met signaling. 6. Gab1 recruitment initiates a pro-tumorgenic PI3K-Akt-mTor signaling pathway. 7. Activation of additional unidentified signaling pathway(s) located downstream from c-Met (hatched arrows) induces constitutive keratinocyte proliferation. Matriptase-induced mTor activation and mitogenic signaling, in combination with other epigenetic and genetic changes ( ras -dependent and ras -independent), causes malignant transformation. The model is synthesized on the basis of data obtained in (7), and the current study.

Article Snippet: The protein concentration was determined by BCA assay (Pierce, Rockford, IL) and 40 μg of total protein (Pierce, Rockford, IL) was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human Matriptase (R&D Systems), rabbit anti-human phospho cMet, phospho-Gab1, or rabbit anti human GAPDH primary antibodies (all Cell Signaling Technology, Beverly, MA).

Techniques: Expressing, Activation Assay, Transformation Assay, Synthesized