Journal: Experimental and Therapeutic Medicine

Article Title: Effects of norepinephrine on colonic tight junction protein expression during heat stress

doi: 10.3892/etm.2021.9865

Figure Lengend Snippet: Expression levels of Occludin, ZO-1, TRPA1 and PAR-2 in the rat distal colon tissue sections in control and heat exposure groups. The figures (A, B) shown are representative of three different animals. Magnification, x200. Black arrows indicate the musca. Red arrows indicate the myenteric plexus. ZO-1, zonula occludens-1; PAR-2, protease-activated receptor 2; TRPA1, transient receptor potential ankyrin 1 channel; CM, circular muscle; LM, longitudinal muscle.

Article Snippet: The cells were then incubated with primary antibodies against PAR-2 (1:300; cat. no. bs-1178R; BIOSS), Occludin (1:100; cat. no. ab216327; Abcam), ZO-1 (1:300; cat. no. bs-1329-R; BIOSS) and TRPA1 (1:200; cat. no. ab58844; Abcam) at 4˚C overnight, followed by incubation with FITC-conjugated goat-anti-rabbit secondary antibodies (1:50; cat. no. bs-0295G-FITC; BIOSS) for 1 h at room temperature in the dark after washing in cold PBS.

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of norepinephrine on colonic tight junction protein expression during heat stress

doi: 10.3892/etm.2021.9865

Figure Lengend Snippet: Expression levels of Occludin, ZO-1, PAR-2 and TRPA1 in Caco-2 cells after treatment with NE. (A) Representative western blotting images from four independent experiments. Comparison of the relative expression levels of (B) Occludin, (C) ZO-1, (D) PAR-2 and (E) TRPA1 in colon of control and NE-treated cells. * P<0.05 vs. Control. Data are presented as the mean ± SD. NE, norepinephrine; ZO-1, zonula occludens-1; PAR-2, protease activated receptor 2; TRPA1, transient receptor potential ankyrin 1 channel.

Article Snippet: The cells were then incubated with primary antibodies against PAR-2 (1:300; cat. no. bs-1178R; BIOSS), Occludin (1:100; cat. no. ab216327; Abcam), ZO-1 (1:300; cat. no. bs-1329-R; BIOSS) and TRPA1 (1:200; cat. no. ab58844; Abcam) at 4˚C overnight, followed by incubation with FITC-conjugated goat-anti-rabbit secondary antibodies (1:50; cat. no. bs-0295G-FITC; BIOSS) for 1 h at room temperature in the dark after washing in cold PBS.

Techniques: Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of norepinephrine on colonic tight junction protein expression during heat stress

doi: 10.3892/etm.2021.9865

Figure Lengend Snippet: Immunofluorescence staining of Occludin, ZO-1, PAR-2 and TRPA1 in Caco-2 cells after 10 µM NE application. Staining of (A) Occludin, (B) ZO-1, (C) PAR-2 and (D) TRPA1. Magnification, x400. NE, norepinephrine; ZO-1, zonula occludens-1; PAR-2, protease-activated receptor 2; TRPA1, transient receptor potential ankyrin 1 channel.

Article Snippet: The cells were then incubated with primary antibodies against PAR-2 (1:300; cat. no. bs-1178R; BIOSS), Occludin (1:100; cat. no. ab216327; Abcam), ZO-1 (1:300; cat. no. bs-1329-R; BIOSS) and TRPA1 (1:200; cat. no. ab58844; Abcam) at 4˚C overnight, followed by incubation with FITC-conjugated goat-anti-rabbit secondary antibodies (1:50; cat. no. bs-0295G-FITC; BIOSS) for 1 h at room temperature in the dark after washing in cold PBS.

Techniques: Immunofluorescence, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of norepinephrine on colonic tight junction protein expression during heat stress

doi: 10.3892/etm.2021.9865

Figure Lengend Snippet: Expression levels of Occludin, ZO-1, TRPA1 and PAR-2 in the rat distal colon tissue sections in control and heat exposure groups. The figures (A, B) shown are representative of three different animals. Magnification, x200. Black arrows indicate the musca. Red arrows indicate the myenteric plexus. ZO-1, zonula occludens-1; PAR-2, protease-activated receptor 2; TRPA1, transient receptor potential ankyrin 1 channel; CM, circular muscle; LM, longitudinal muscle.

Article Snippet: The cells were then incubated with primary antibodies against PAR-2 (1:300; cat. no. bs-1178R; BIOSS), Occludin (1:100; cat. no. ab216327; Abcam), ZO-1 (1:300; cat. no. bs-1329-R; BIOSS) and TRPA1 (1:200; cat. no. ab58844; Abcam) at 4˚C overnight, followed by incubation with FITC-conjugated goat-anti-rabbit secondary antibodies (1:50; cat. no. bs-0295G-FITC; BIOSS) for 1 h at room temperature in the dark after washing in cold PBS.

Techniques: Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of norepinephrine on colonic tight junction protein expression during heat stress

doi: 10.3892/etm.2021.9865

Figure Lengend Snippet: Expression levels of Occludin, ZO-1, PAR-2 and TRPA1 in Caco-2 cells after treatment with NE. (A) Representative western blotting images from four independent experiments. Comparison of the relative expression levels of (B) Occludin, (C) ZO-1, (D) PAR-2 and (E) TRPA1 in colon of control and NE-treated cells. * P<0.05 vs. Control. Data are presented as the mean ± SD. NE, norepinephrine; ZO-1, zonula occludens-1; PAR-2, protease activated receptor 2; TRPA1, transient receptor potential ankyrin 1 channel.

Article Snippet: The cells were then incubated with primary antibodies against PAR-2 (1:300; cat. no. bs-1178R; BIOSS), Occludin (1:100; cat. no. ab216327; Abcam), ZO-1 (1:300; cat. no. bs-1329-R; BIOSS) and TRPA1 (1:200; cat. no. ab58844; Abcam) at 4˚C overnight, followed by incubation with FITC-conjugated goat-anti-rabbit secondary antibodies (1:50; cat. no. bs-0295G-FITC; BIOSS) for 1 h at room temperature in the dark after washing in cold PBS.

Techniques: Expressing, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of norepinephrine on colonic tight junction protein expression during heat stress

doi: 10.3892/etm.2021.9865

Figure Lengend Snippet: Immunofluorescence staining of Occludin, ZO-1, PAR-2 and TRPA1 in Caco-2 cells after 10 µM NE application. Staining of (A) Occludin, (B) ZO-1, (C) PAR-2 and (D) TRPA1. Magnification, x400. NE, norepinephrine; ZO-1, zonula occludens-1; PAR-2, protease-activated receptor 2; TRPA1, transient receptor potential ankyrin 1 channel.

Article Snippet: The cells were then incubated with primary antibodies against PAR-2 (1:300; cat. no. bs-1178R; BIOSS), Occludin (1:100; cat. no. ab216327; Abcam), ZO-1 (1:300; cat. no. bs-1329-R; BIOSS) and TRPA1 (1:200; cat. no. ab58844; Abcam) at 4˚C overnight, followed by incubation with FITC-conjugated goat-anti-rabbit secondary antibodies (1:50; cat. no. bs-0295G-FITC; BIOSS) for 1 h at room temperature in the dark after washing in cold PBS.

Techniques: Immunofluorescence, Staining

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 expression level in lung cancer cells transfected either with pcDNA3-PAR2 or PAR2 shRNA. (A) PAR2 expression level after A549 cells were transfected with pcDNA3-PAR2; (B) PAR2 expression level after H1299 cells were transfected with pcDNA3-PAR2; (C) PAR2 expression level after A549 cells were transfected with PAR2 shRNA; (D) PAR2 expression level after H1299 cells were transfected with PAR2 shRNA.

Article Snippet: Human PAR2 sandwich enzyme-linked immunosorbent assay (ELISA) kit (Novus Biologicals, USA) was used to detect serum PAR2 level according to the manufacture’s instructions.

Techniques: Expressing, Transfection, shRNA

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: Overexpression PAR2 promoted growth of lung cancer cells with or without paclitaxel. (A–D) The growth of A549 cells with or without paclitaxel after transfection with pcDNA3-PAR2; (E–H) The growth of H1299 cells with or without paclitaxel after transfection with pcDNA3-PAR2.

Article Snippet: Human PAR2 sandwich enzyme-linked immunosorbent assay (ELISA) kit (Novus Biologicals, USA) was used to detect serum PAR2 level according to the manufacture’s instructions.

Techniques: Over Expression, Transfection

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: Knockdown PAR2 decreased growth of lung cancer cells with or without paclitaxel. (A–D) The growth of A549 cells with or without paclitaxel after transfection with PAR2 shRNA; (E–H) The growth of H1299 cells with or without paclitaxel after transfection with PAR2 shRNA.

Article Snippet: Human PAR2 sandwich enzyme-linked immunosorbent assay (ELISA) kit (Novus Biologicals, USA) was used to detect serum PAR2 level according to the manufacture’s instructions.

Techniques: Knockdown, Transfection, shRNA

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 inhibited paclitaxel-associated apoptosis in lung cancer cells. (A) Caspase 3/7 activity in A549 cell transfected with pcDNA3-PAR2; (B) Caspase 3/7 activity in H1299 cell transfected with pcDNA3-PAR2; (C–F) Bcl-2 and BAX expression in A549 and H1299 cells.

Article Snippet: Human PAR2 sandwich enzyme-linked immunosorbent assay (ELISA) kit (Novus Biologicals, USA) was used to detect serum PAR2 level according to the manufacture’s instructions.

Techniques: Activity Assay, Transfection, Expressing

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 played essential roles in invasion and migration of lung cancer cells. (A, B) up-regulation of PAR2 increased migration and invasion of A549 cells. (C, D) down-regulation of PAR2 decreaased migration and invasion of A549 cells. (E, F) up-regulation of PAR2 increased migration and invasion of H1299 cells. (G, H) down-regulation of PAR2 decreased migration and invasion of H1299 cells.

Article Snippet: Human PAR2 sandwich enzyme-linked immunosorbent assay (ELISA) kit (Novus Biologicals, USA) was used to detect serum PAR2 level according to the manufacture’s instructions.

Techniques: Migration

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 altered PTEN/AKT protein expression in lung cancer cells. (A–C) Impact of up-regulation of PAR2 on expression of p-AKT, AKT and PTEN in A549 and H1299 cells. (D–F) Impact of down-regulation of PAR2 on expression of p-AKT, AKT and PTEN in A549 and H1299 cells.

Article Snippet: Human PAR2 sandwich enzyme-linked immunosorbent assay (ELISA) kit (Novus Biologicals, USA) was used to detect serum PAR2 level according to the manufacture’s instructions.

Techniques: Expressing

Journal: Future Science OA

Article Title: PAR2 regulates proliferation, migration of lung cancer and chemotherapy sensitivity by involving PTEN pathway

doi: 10.1080/20565623.2025.2535221

Figure Lengend Snippet: PAR2 expression in human lung cancer tissue. (A) PAR2 levels in different stage of lung cancer tissue and normal lung tissue. (B) Immunohistochemical studies for Ki-67 and PTEN on different levels of PAR2 in lung tissue.

Article Snippet: Human PAR2 sandwich enzyme-linked immunosorbent assay (ELISA) kit (Novus Biologicals, USA) was used to detect serum PAR2 level according to the manufacture’s instructions.

Techniques: Expressing, Immunohistochemical staining

Journal: Frontiers in Oncology

Article Title: The protease-activated receptors are expressed in glioblastoma and differentially modulate adherent versus stem-like growth of LN-18 GBM cells

doi: 10.3389/fonc.2025.1582996

Figure Lengend Snippet: PAR1–4 mRNA expression in GBM specimen and its association with patients´ overall survival time. (A) Comparison of PAR1, PAR2, PAR3 and PAR4 mRNA expression in non-malignant brain (NMB, n=7) and all analyzed GBM samples (both primary and relapsed GBM, n=118). (B) Subdivision of GBM specimen in primary GBM (prGBM, n=78), first (1 st , n=33) and second (2 nd , n=7) relapses and comparison with NMB. (A+B) Gene expression was measured by qPCR. Each mRNA level of the target genes (PAR1-4) was normalized to the mean of GAPDH and β-actin using the 2 -ΔΔct method. Data are shown as scatter plots representing the median as horizontal bars. Mann Whitney U test, *p < 0.05 and ***p < 0.001 for (A) and OneWay ANOVA/Kruskal Wallis test with Dunn’s Multiple Comparison Test, *p < 0.05, **p < 0.01 and *** p < 0.001 for (B) . (C) Kaplan Meier survival analyses of PAR1–4 mRNA expression in GBM patients. Association of the relative mRNA expression of each single PAR receptor with the survival time of patients with primary GBM. The patients were divided into two subgroups depending on the median gene expression. No significant association was found.

Article Snippet: As specific PAR antagonists the following compounds were used: PAR1 antagonist RWJ, PAR2 antagonist FSLLRY-NH2, and PAR4 antagonist tcY-NH2 (all from Tocris Bioscience).

Techniques: Expressing, Comparison, Gene Expression, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: The protease-activated receptors are expressed in glioblastoma and differentially modulate adherent versus stem-like growth of LN-18 GBM cells

doi: 10.3389/fonc.2025.1582996

Figure Lengend Snippet: Impact of PAR subtype specific inhibitors and agonists on viability of LN-18 GBM cells. Adherent and neurospheric LN-18 cells of passage 1 or 3 were treated for 48 or 72h with the respective compounds followed by measurement of cell viability using the Resazurin assay. (A+B) Treatment of adherent (A) and neurospheric (B) LN-18 cells with inhibitors of PAR1 (RWJ), PAR2 (FSLLRY) and PAR4 (tcY) (each 5 and 20 µM), n=3-4. (C+D) Incubation of adherent (C) and neurospheric (D) LN-18 cells with Thrombin (TB, 30 U/ml), Factor Xa (FXa, 30 nM) or PAR subtype specific agonists (AP1 to AP4, each 100 and 200 µM), n=3-4. OneWay ANOVA Dunnett’s Multiple Comparison Test, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. Con.

Article Snippet: As specific PAR antagonists the following compounds were used: PAR1 antagonist RWJ, PAR2 antagonist FSLLRY-NH2, and PAR4 antagonist tcY-NH2 (all from Tocris Bioscience).

Techniques: Resazurin Assay, Incubation, Comparison

Journal: Biomolecules

Article Title: Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis

doi: 10.3390/biom12071003

Figure Lengend Snippet: Estimation of KLK6 expression in colon carcinoma epithelial cells.

Article Snippet: KLK6 also cleaved efficiently the substrates mimicking PAR1 and PAR2 N-terminal peptides (Data not shown); human α-thrombin (3000 U/mg) from Stago BNL (Lille, France); pancreatic porcine trypsin (16,000 U/mg) from Sigma Aldrich (Saint-Quentin-Fallavier, France); pyromycin, DMEM, RPMI 1640, and HAM’s F12 medium were purchased from Life Techonologies (Cergy-Pontoise, France); human KLK6 polyclonal antibody from Boster (Pleasanton, CA, USA), monoclonal anti-PAR2 (SAM11) (# SC-13504)(Clinisciences, Nanterre, France) and anti-monoclonal PAR1 (WEDE15) (#PN IM2085°) from Beckman Coulter (Marseille, France) and phospho-specific antibodies to ERK1/2 (Thr202/Tyr204) (#9106) from Cell Signaling Technologies (Beverly, MA, USA); anti-ERK1/2 (#SC-94) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); peroxidase-conjugated anti-mouse (#115-035-068) and anti-rabbit (#111-035-144) antibodies were from Jackson Immuno-Research (West Grove, PA, USA).

Techniques: Expressing, Staining

Journal: Biomolecules

Article Title: Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis

doi: 10.3390/biom12071003

Figure Lengend Snippet: Expression of KLK6 in human colon cancer cell lines. ( A ) Two micrograms of total RNA were reverse transcribed and PCR-amplified with KLK6 or GAPDH primers as described in Material and Methods. A major single PCR-amplified product of the predicted size (774 bp) for KLK6 was visualized after electrophoresis on a 2% agarose gel. GADPH was used as an internal control. Normal isolated epithelial cells do not express KLK 6 mRNA. Note that KLK 6 is present in SW620, a cell line from a lymph node of a primary adenocarcinoma from which SW480 was derived. ( B ) The amount of mRNA expression was quantified by densitometry of bands in comparison to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Densitometry of mRNA bands were quantified from three independent PCR experiments presented as mean ± SEM. ( C ) Immunodetection of kallikrein-related peptidases 6 by colon cancer cell lines: Supernatants were collected from colon cancer cells in culture and KLK6 expression was estimated by sandwich-type ELISA (see Material and Methods). Protein values represent the mean concentration of KLK6 (µg/L) secreted by 10 6 cells, which were cultured for 24 h. Inset: shows confocal microscopic immunocytochemical localization of KLK6 in HT-29 cells (Magnification X630). Arrows show cytoplasmic, perinuclear staining of KLK6.

Article Snippet: KLK6 also cleaved efficiently the substrates mimicking PAR1 and PAR2 N-terminal peptides (Data not shown); human α-thrombin (3000 U/mg) from Stago BNL (Lille, France); pancreatic porcine trypsin (16,000 U/mg) from Sigma Aldrich (Saint-Quentin-Fallavier, France); pyromycin, DMEM, RPMI 1640, and HAM’s F12 medium were purchased from Life Techonologies (Cergy-Pontoise, France); human KLK6 polyclonal antibody from Boster (Pleasanton, CA, USA), monoclonal anti-PAR2 (SAM11) (# SC-13504)(Clinisciences, Nanterre, France) and anti-monoclonal PAR1 (WEDE15) (#PN IM2085°) from Beckman Coulter (Marseille, France) and phospho-specific antibodies to ERK1/2 (Thr202/Tyr204) (#9106) from Cell Signaling Technologies (Beverly, MA, USA); anti-ERK1/2 (#SC-94) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); peroxidase-conjugated anti-mouse (#115-035-068) and anti-rabbit (#111-035-144) antibodies were from Jackson Immuno-Research (West Grove, PA, USA).

Techniques: Expressing, Reverse Transcription, Amplification, Electrophoresis, Agarose Gel Electrophoresis, Control, Isolation, Derivative Assay, Comparison, Immunodetection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Staining

Journal: Biomolecules

Article Title: Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis

doi: 10.3390/biom12071003

Figure Lengend Snippet: KLK6 induces loss of PAR1 and PAR2 from the surface of HT-29 cells and initiates calcium signaling via PAR2. ( A ) Immunofluorescence detection of PAR1 and PAR2 in HT-29 cells pre-treated with KLK6 (20 nmol/L), or with vehicle (control) for 15 min at 37 °C. Cells were fixed using 2% paraformaldehyde and immunostained with a PAR2 monoclonal antibody or PAR1 monoclonal antibody. Results are representative of two independent experiments (Magnification ×630). ( B , C ) KLK6 initiates intracellular Ca 2+ mobilization. HT-29 cells were loaded for 60 min at 37 °C using Fura 2/AM. ( B ) HT-29 cells were challenged first with AP2 (SLIGKV-NH 2 , 100 µmol/L)) and AP1 (TFFLLR-NH 2 , 100 µmol/L). ( C ) Cells were first challenged with KLK6 (1 µmol/L) followed by a second challenge with activating peptide AP2 (100 µmol/L) and subsequent challenge with AP1 (100 µmol/L). Note the AP2 response showed a reduction in response, whereas cells were still responsive to AP1. Administration of compounds is indicated with the corresponding arrows.

Article Snippet: KLK6 also cleaved efficiently the substrates mimicking PAR1 and PAR2 N-terminal peptides (Data not shown); human α-thrombin (3000 U/mg) from Stago BNL (Lille, France); pancreatic porcine trypsin (16,000 U/mg) from Sigma Aldrich (Saint-Quentin-Fallavier, France); pyromycin, DMEM, RPMI 1640, and HAM’s F12 medium were purchased from Life Techonologies (Cergy-Pontoise, France); human KLK6 polyclonal antibody from Boster (Pleasanton, CA, USA), monoclonal anti-PAR2 (SAM11) (# SC-13504)(Clinisciences, Nanterre, France) and anti-monoclonal PAR1 (WEDE15) (#PN IM2085°) from Beckman Coulter (Marseille, France) and phospho-specific antibodies to ERK1/2 (Thr202/Tyr204) (#9106) from Cell Signaling Technologies (Beverly, MA, USA); anti-ERK1/2 (#SC-94) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); peroxidase-conjugated anti-mouse (#115-035-068) and anti-rabbit (#111-035-144) antibodies were from Jackson Immuno-Research (West Grove, PA, USA).

Techniques: Immunofluorescence, Control

Journal: Biomolecules

Article Title: Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis

doi: 10.3390/biom12071003

Figure Lengend Snippet: Dose-dependent activation of p42/p44 MAP-Kinase phosphorylation by KLK6. ( A ) Quiescent HT-29 cells were stimulated with the indicated concentrations of KLK6, thrombin (0.01 µmol/L), trypsin (0.01 µmol/L), with 100 µM of TFLLR-NH 2 (AP1, a PAR1 agonist), 100 µM SLIGKV-NH 2 (AP2, a PAR2 agonist) or with vehicle (control) for 5 min. ( B ) Quiescent HCT116 cells were stimulated with the indicated concentrations of KLK6, with 100 µM of TFLLR-NH 2 (AP1, a PAR1 agonist), with 100 µM SLIGKV-NH 2 (AP2, a PAR2 agonist) or with vehicle (control) for 5 min. To confirm equal protein loading, the membranes were stripped and incubated with p42/p44 MAP-Kinase antibody. p42/p44 MAPKinase protein seems to be reduced in PARs agonist-stimulated samples, possibly because the high signal with the anti-phopsho-ERK1/2 (Thr202/Tyr204) antibody prevents subsequent anti-ERK1/2 antibody binding to the ERK1/2 epitope as observed previously . Densitometric analysis of the phospo-p42/p44 MAP-Kinase divided by total amount of p42/p44 MAP-Kinase is represented in lower panels. Results are representative of two separate experiments.

Article Snippet: KLK6 also cleaved efficiently the substrates mimicking PAR1 and PAR2 N-terminal peptides (Data not shown); human α-thrombin (3000 U/mg) from Stago BNL (Lille, France); pancreatic porcine trypsin (16,000 U/mg) from Sigma Aldrich (Saint-Quentin-Fallavier, France); pyromycin, DMEM, RPMI 1640, and HAM’s F12 medium were purchased from Life Techonologies (Cergy-Pontoise, France); human KLK6 polyclonal antibody from Boster (Pleasanton, CA, USA), monoclonal anti-PAR2 (SAM11) (# SC-13504)(Clinisciences, Nanterre, France) and anti-monoclonal PAR1 (WEDE15) (#PN IM2085°) from Beckman Coulter (Marseille, France) and phospho-specific antibodies to ERK1/2 (Thr202/Tyr204) (#9106) from Cell Signaling Technologies (Beverly, MA, USA); anti-ERK1/2 (#SC-94) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); peroxidase-conjugated anti-mouse (#115-035-068) and anti-rabbit (#111-035-144) antibodies were from Jackson Immuno-Research (West Grove, PA, USA).

Techniques: Activation Assay, Phospho-proteomics, Control, Incubation, Binding Assay

Journal: Biomolecules

Article Title: Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis

doi: 10.3390/biom12071003

Figure Lengend Snippet: The effect of KLK6 knockdown on colony formation. ( A ) HCT116-control shRNA and HCT116-KLK6 shRNA were plated as described in Materials and Methods and incubated for 12 days. Silencing KLK6 induced a strong decrease in colon cancer colony formation. Representative images were captured and quantified using an Azure™ Imaging Systems. ( B ) Analysis of colony formation rates is shown as percentage of colonies from three independent experiments, each performed in duplicate. Data are shown as mean ± SEM. *** p < 0.001.

Article Snippet: KLK6 also cleaved efficiently the substrates mimicking PAR1 and PAR2 N-terminal peptides (Data not shown); human α-thrombin (3000 U/mg) from Stago BNL (Lille, France); pancreatic porcine trypsin (16,000 U/mg) from Sigma Aldrich (Saint-Quentin-Fallavier, France); pyromycin, DMEM, RPMI 1640, and HAM’s F12 medium were purchased from Life Techonologies (Cergy-Pontoise, France); human KLK6 polyclonal antibody from Boster (Pleasanton, CA, USA), monoclonal anti-PAR2 (SAM11) (# SC-13504)(Clinisciences, Nanterre, France) and anti-monoclonal PAR1 (WEDE15) (#PN IM2085°) from Beckman Coulter (Marseille, France) and phospho-specific antibodies to ERK1/2 (Thr202/Tyr204) (#9106) from Cell Signaling Technologies (Beverly, MA, USA); anti-ERK1/2 (#SC-94) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); peroxidase-conjugated anti-mouse (#115-035-068) and anti-rabbit (#111-035-144) antibodies were from Jackson Immuno-Research (West Grove, PA, USA).

Techniques: Knockdown, Control, shRNA, Incubation, Imaging

Journal: Biomolecules

Article Title: Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis

doi: 10.3390/biom12071003

Figure Lengend Snippet: Effect of KLK6 knock down on cell adhesion. The adhesion assay was performed by seeding HCT116-KLK6-KO and HCT116-control cells (10 5 cells each) in 96-well plates coated with type I collagen (Col-I), type II collagen (Col-II), type IV collagen (Col-IV), fibronectin (FN), laminin (LN), tenascin (TN), or BSA as a negative control. Cells were incubated for 2 h. Non-adherent cells were washed three times with PBS, the attached cells were then stained using the ECM Cell Adhesion Array Kit and quantified by absorbance readings in a microplate reader at 570 nm. Means of three independent experiments are presented. Data are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: KLK6 also cleaved efficiently the substrates mimicking PAR1 and PAR2 N-terminal peptides (Data not shown); human α-thrombin (3000 U/mg) from Stago BNL (Lille, France); pancreatic porcine trypsin (16,000 U/mg) from Sigma Aldrich (Saint-Quentin-Fallavier, France); pyromycin, DMEM, RPMI 1640, and HAM’s F12 medium were purchased from Life Techonologies (Cergy-Pontoise, France); human KLK6 polyclonal antibody from Boster (Pleasanton, CA, USA), monoclonal anti-PAR2 (SAM11) (# SC-13504)(Clinisciences, Nanterre, France) and anti-monoclonal PAR1 (WEDE15) (#PN IM2085°) from Beckman Coulter (Marseille, France) and phospho-specific antibodies to ERK1/2 (Thr202/Tyr204) (#9106) from Cell Signaling Technologies (Beverly, MA, USA); anti-ERK1/2 (#SC-94) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); peroxidase-conjugated anti-mouse (#115-035-068) and anti-rabbit (#111-035-144) antibodies were from Jackson Immuno-Research (West Grove, PA, USA).

Techniques: Knockdown, Cell Adhesion Assay, Control, Negative Control, Incubation, Staining

Journal: Biomolecules

Article Title: Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis

doi: 10.3390/biom12071003

Figure Lengend Snippet: Effect of KLK6 silencing on colon cancer spheroid formation. Spheres were generated by growing cells at a clonal density of 1000 cells/mL in neural crest cells culture conditions as described in Material and Methods. Cells were dispensed in low-adherent plates and medium was changed twice a week. Spheres were allowed to grow over the course of 12 days. Spheres were counted and measured every 2–3 days under the microscope. ( A ) Upper panel: KLK6 silencing results in altered cell morphology. Lower panel: representative images of spheroids formed by HCT116-KLK6 shRNA and HCT116-control shRNA cells randomly taken at day 6 after subculture. ( B ) Spheroids number formed by HCT116-KLK6 shRNA and HCT116-control shRNA cells. Data are shown as mean ± SEM of three independent experiments each performed in duplicate. ** p < 0.01.

Article Snippet: KLK6 also cleaved efficiently the substrates mimicking PAR1 and PAR2 N-terminal peptides (Data not shown); human α-thrombin (3000 U/mg) from Stago BNL (Lille, France); pancreatic porcine trypsin (16,000 U/mg) from Sigma Aldrich (Saint-Quentin-Fallavier, France); pyromycin, DMEM, RPMI 1640, and HAM’s F12 medium were purchased from Life Techonologies (Cergy-Pontoise, France); human KLK6 polyclonal antibody from Boster (Pleasanton, CA, USA), monoclonal anti-PAR2 (SAM11) (# SC-13504)(Clinisciences, Nanterre, France) and anti-monoclonal PAR1 (WEDE15) (#PN IM2085°) from Beckman Coulter (Marseille, France) and phospho-specific antibodies to ERK1/2 (Thr202/Tyr204) (#9106) from Cell Signaling Technologies (Beverly, MA, USA); anti-ERK1/2 (#SC-94) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); peroxidase-conjugated anti-mouse (#115-035-068) and anti-rabbit (#111-035-144) antibodies were from Jackson Immuno-Research (West Grove, PA, USA).

Techniques: Generated, Microscopy, shRNA, Control

Journal: Biomolecules

Article Title: Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis

doi: 10.3390/biom12071003

Figure Lengend Snippet: Representative immunostaining of the KLK6 in paraffin sections of colonic tissues from patients with adenocarcinoma. ( A ) KLK6 immunoreactivity is absent in ‘normal-appearing’ colonic mucosa very distant (more than 10 cm) from a colon adenocarcinoma ( B ) Immunostaining for KLK6 in the dysplastic colonic mucosa from the same patient as in ( A ) is seen. ( C – F ) Moderate to strong positive immunoreactivity in the adenocarcinomas of four different patients. KLK6 immunoreactivity appeared to be distinguishable into a predominantly apical/cytoplasmic pattern (arrows). Bar = 100 µm ( A – F ), 10 µm ( D ).

Article Snippet: KLK6 also cleaved efficiently the substrates mimicking PAR1 and PAR2 N-terminal peptides (Data not shown); human α-thrombin (3000 U/mg) from Stago BNL (Lille, France); pancreatic porcine trypsin (16,000 U/mg) from Sigma Aldrich (Saint-Quentin-Fallavier, France); pyromycin, DMEM, RPMI 1640, and HAM’s F12 medium were purchased from Life Techonologies (Cergy-Pontoise, France); human KLK6 polyclonal antibody from Boster (Pleasanton, CA, USA), monoclonal anti-PAR2 (SAM11) (# SC-13504)(Clinisciences, Nanterre, France) and anti-monoclonal PAR1 (WEDE15) (#PN IM2085°) from Beckman Coulter (Marseille, France) and phospho-specific antibodies to ERK1/2 (Thr202/Tyr204) (#9106) from Cell Signaling Technologies (Beverly, MA, USA); anti-ERK1/2 (#SC-94) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); peroxidase-conjugated anti-mouse (#115-035-068) and anti-rabbit (#111-035-144) antibodies were from Jackson Immuno-Research (West Grove, PA, USA).

Techniques: Immunostaining

Journal: Biomolecules

Article Title: Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis

doi: 10.3390/biom12071003

Figure Lengend Snippet: Immunodetection of KLK 6 in ascitic peritoneal metastasis from human colorectal cancer. Ascitic fluids were collected from peritoneal metastasis from human colorectal cancer (Colon-PM), (Gastric-PM) or from non-malignant liver diseases (NM Liver disease) and KLK6 expression was estimated by a sandwich-type ELISA (see Material and Methods). Protein values represent the mean concentration of KLK6 (µg/L). Data are shown as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: KLK6 also cleaved efficiently the substrates mimicking PAR1 and PAR2 N-terminal peptides (Data not shown); human α-thrombin (3000 U/mg) from Stago BNL (Lille, France); pancreatic porcine trypsin (16,000 U/mg) from Sigma Aldrich (Saint-Quentin-Fallavier, France); pyromycin, DMEM, RPMI 1640, and HAM’s F12 medium were purchased from Life Techonologies (Cergy-Pontoise, France); human KLK6 polyclonal antibody from Boster (Pleasanton, CA, USA), monoclonal anti-PAR2 (SAM11) (# SC-13504)(Clinisciences, Nanterre, France) and anti-monoclonal PAR1 (WEDE15) (#PN IM2085°) from Beckman Coulter (Marseille, France) and phospho-specific antibodies to ERK1/2 (Thr202/Tyr204) (#9106) from Cell Signaling Technologies (Beverly, MA, USA); anti-ERK1/2 (#SC-94) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); peroxidase-conjugated anti-mouse (#115-035-068) and anti-rabbit (#111-035-144) antibodies were from Jackson Immuno-Research (West Grove, PA, USA).

Techniques: Immunodetection, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay