Journal: Cells

Article Title: KLK6/PAR1 Axis Promotes Tumor Growth and Metastasis by Regulating Cross-Talk between Tumor Cells and Macrophages.

doi: 10.3390/cells11244101

Figure Lengend Snippet: Figure 6. KLK6 stimulated TNF-α production in macrophages via PAR1. (A–D) RAW 264.7 cells were transfected with control siRNA, two PAR1 siRNAs, or two PAR2 siRNAs. (A) PAR1 and PAR2 mRNA levels in the indicated siRNAs-transfected cells were measured by RT-PCR. (B) The level of secreted TNF-α in the supernatants of the indicated siRNAs-transfected cells was analyzed by ELISA. (C,D) B16F10 and LLC cells were treated with the CMs of the indicated siRNA-transfected RAW 264.7 cells for 24 h. The mRNA (C) and secreted protein (D) of CXCL1 in B16F10 and LLC cells treated with the CMs of indicated RAW 264.7 cells were analyzed by RT-PCR and ELISA, respectively. (E–G) RAW 264.7 cells were treated with DMSO (as control), a PAR1 antagonist, or a PAR2 antagonist for 24 h. (E) The level of secreted TNF-α in the supernatants of the indicated RAW 264.7 cells was analyzed by ELISA. (F) The level of CXCL1 mRNA in the B16F10 and LLC cells treated with the CMs of indicated RAW 264.7 cells was analyzed by RT-PCR. (G) The level of secreted CXCL1 in the supernatants of B16F10 cells treated with the CMs of the indicated RAW 264.7 cells was measured by ELISA. Statistical significance was determined by the Student’s t-test. * p < 0.05 and ** p < 0.01. Data are representative of three experiments.

Article Snippet: CXCL1 neutralizing antibody (R&D Systems, Minneapolis, MN, USA, MAB453), CXCL1 (R&D Systems, AF-453), PAR1 antagonist (Med-ChemExpress, Monmouth Junction, NJ, USA, SCH 79797), and PAR2 antagonist (Axon-Medchem, Reston, VA, USA, Axon 1622) were purchased from R&D Systems, MedChemExpress and AxonMedchem, respectively. β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc47778), Ki67 (Santa Cruz, sc-550609), GFP (Santa Cruz, sc-9996), PAR1 (Novus Biologicals, Novus, Centennial, CO, USA, NBP1-71770), and PAR2 (Abcam, Cambridge, UK, ab180953) antibodies were obtained from Santa Cruz Biotechnology, Novus Biologicals and Abcam, respectively. β-actin was used to normalize the level of proteins.

Techniques: Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: KLK6/PAR1 Axis Promotes Tumor Growth and Metastasis by Regulating Cross-Talk between Tumor Cells and Macrophages.

doi: 10.3390/cells11244101

Figure Lengend Snippet: Figure 7. Recombinant KLK6 promoted macrophage-derived TNF-α production via PAR1 in KLK6−/−BMDMs. (A) KLK6−/−BMDMs were treated with rKLK6 at the indicated concen- trations for 24 h. The secreted TNF-α levels were measured by ELISA. (B,C) B16F10 cells were treated with the CMs of KLK6−/−BMDMs treated with rKLK6 at the indicated conditions for 24 h. The levels of CXCL1 mRNA (B) and secreted CXCL1 protein (C) were analyzed by RT-PCR and ELISA, respectively. (D) KLK6−/−BMDMs were treated with rKLK6 in the presence or absence of the PAR1 antagonist (100 nM). The secreted TNF-α levels were measured by ELISA. (E) B16F10 cells were treated with the CMs of indicated KLK6−/−BMDMs for 24 h. The secreted CXCL1 levels were measured by ELISA. ** p < 0.01 and * p < 0.05 (Student’s t-test). Data are representative of three experiments.

Article Snippet: CXCL1 neutralizing antibody (R&D Systems, Minneapolis, MN, USA, MAB453), CXCL1 (R&D Systems, AF-453), PAR1 antagonist (Med-ChemExpress, Monmouth Junction, NJ, USA, SCH 79797), and PAR2 antagonist (Axon-Medchem, Reston, VA, USA, Axon 1622) were purchased from R&D Systems, MedChemExpress and AxonMedchem, respectively. β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc47778), Ki67 (Santa Cruz, sc-550609), GFP (Santa Cruz, sc-9996), PAR1 (Novus Biologicals, Novus, Centennial, CO, USA, NBP1-71770), and PAR2 (Abcam, Cambridge, UK, ab180953) antibodies were obtained from Santa Cruz Biotechnology, Novus Biologicals and Abcam, respectively. β-actin was used to normalize the level of proteins.

Techniques: Recombinant, Derivative Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Cells

Article Title: KLK6/PAR1 Axis Promotes Tumor Growth and Metastasis by Regulating Cross-Talk between Tumor Cells and Macrophages.

doi: 10.3390/cells11244101

Figure Lengend Snippet: Figure 8. PAR1 inhibitor reduced rKLK6-mediated tumor growth and lung metastasis. (A–E) B16F10 cells (1 × 106) were subcutaneously injected into KLK6−/−mice. After 1 h, DMSO (control), rKLK6 (10 mg/kg), or rKLK6 plus PAR1 (5 mM/kg) were injected every other day for 10 days. (A) Representative photograph of the tumors. The tumor volume (B) and tumor weight (C) were measured as described in the Materials and Methods. The levels of serum TNF-α (D) and CXCL1 (E) were measured by ELISA. (F,J) B16F10 cells (5 × 105) were injected into the tail vein of WT mice. After 1 h, DMSO (control), rKLK6 (10 mg/kg), or rKLK6 plus PAR1 (5 mM/kg) were injected every other day for 10 days. (F) Representative photograph of metastatic lung nodules. The number of metastatic lung nodules (G) were calculated as described in the Materials and Methods. (H) The weight of lungs with metastasized nodules:body weight. The levels of serum TNF-α (I) and CXCL1 (J) were analyzed by ELISA. ** p < 0.01 and * p < 0.05 (Student’s t-test). Data are representative of three experiments.

Article Snippet: CXCL1 neutralizing antibody (R&D Systems, Minneapolis, MN, USA, MAB453), CXCL1 (R&D Systems, AF-453), PAR1 antagonist (Med-ChemExpress, Monmouth Junction, NJ, USA, SCH 79797), and PAR2 antagonist (Axon-Medchem, Reston, VA, USA, Axon 1622) were purchased from R&D Systems, MedChemExpress and AxonMedchem, respectively. β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc47778), Ki67 (Santa Cruz, sc-550609), GFP (Santa Cruz, sc-9996), PAR1 (Novus Biologicals, Novus, Centennial, CO, USA, NBP1-71770), and PAR2 (Abcam, Cambridge, UK, ab180953) antibodies were obtained from Santa Cruz Biotechnology, Novus Biologicals and Abcam, respectively. β-actin was used to normalize the level of proteins.

Techniques: Injection, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Bar graph showing effects of PAR1 inhibitors on rat oligodendrocyte precursor cells (OPC) cultured in serum-free defined media. Cell death was measured by LDH release (optical density units; mean ± SEM). At high concentrations both SCH79797 and BMS20061 were toxic to the cells (*p < 0.05).

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Cell Culture

Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Bar graph showing effects of plasma alone and in combination with PAR1 inhibitors on rat oligodendrocyte precursor cells (OPC) in culture. Cell death was measured by LDH release (optical density units; mean ± SEM). Plasma at 1/250 concentration added to the defined culture media was toxic to OPC as indicated by increased LDH release. There was no evidence for protection with SCH79797 and BMS20061 at any dose. Control values (no plasma) were significantly less than all groups (*p < 0.05) and there were no statistically significant differences between treatment groups (all comparisons p > 0.5).

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Clinical Proteomics, Concentration Assay, Control

Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Cell proliferation in immature mouse brain following periventricular blood injection. A - Ki67 nuclear immunoreactivity (brown stained nuclei; hematoxylin counterstain) is prominent in the frontal periventricular subventricular zone (SVZ, arrow) of control (i.e. no blood injection) mice from all three genotypes (this example is wild type). B – Following blood injection, some of which extends into the frontal horn of the lateral ventricle (arrow), Ki67 immunoreactivity in the SVZ is reduced (this example is a PAR1 knockout). C – Quantitative analysis (ANOVA) shows the proportion of Ki67 immunoreactive cells in the SVZ was significantly reduced in the 24-hour heterozygous (Het; *p = 0.0050) and the 48-hour wild type (WT; *p = 0.0054) mice; the reduction also approached significance in the 48-hour knockout (KO; p = 0.0577) in comparison to controls. Bar = 100 μm.

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Injection, Staining, Control, Knock-Out, Comparison

Journal: Journal of Negative Results in Biomedicine

Article Title: Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma

doi: 10.1186/s12952-014-0022-4

Figure Lengend Snippet: Cell death in immature mouse brain following periventricular blood injection. A - Activated caspase 3 immunoreactivity is extremely rare in the SVZ of control mice (brown stained cells; hematoxylin counterstain) (arrow; this example is wild type). B – Following blood injection, activated caspase 3 immunoreactivity is prominent in the striatum (arrow) but very rare in the SVZ (this example is a PAR1 knockout). C – Quantitative analysis shows a statistically significant increase in caspase 3 immunoreactive cells in the striatum of most groups in comparison to intact control (p < 0.007), but there were no significant differences between the blood-injected groups (all p > 0.15; Wilcoxon method). All micrographs were taken at 400× magnification. Bar = 100 μm.

Article Snippet: Four hours later the media was changed to serum-free Sato’s medium with added 10 μM bromodeoxyuridine (BrdU Sigma B-5002) with or without added PAR1 antagonists SCH-79797 dihydrochloride (Tocris, Ellisville, MO, USA) or BMS-200261 (Sigma).

Techniques: Injection, Control, Staining, Knock-Out, Comparison