Journal: International Journal of Molecular Sciences

Article Title: GB83, an Agonist of PAR2 with a Unique Mechanism of Action Distinct from Trypsin and PAR2-AP

doi: 10.3390/ijms231810631

Figure Lengend Snippet: Effect of GB83 on PAR-2-mediated increase in intracellular calcium levels in HT-29 cells. ( A ) Inhibitory effect of GB83 on PAR2-mediated intracellular calcium increase. HT-29 cells were pretreated with GB83 at indicated concentrations and AZ3451 (1 µM) for 15 min before PAR2 stimulation by PAR2-AP (30 µM). ( B ) Summary of dose–responses (mean ± S.E., n = 6). ( C – E ) Intracellular calcium levels were increased by the indicated concentrations of trypsin, GB83, and PAR2-AP in HT-29 cells. Intracellular calcium increase induced by trypsin (30 U/mL), GB83 (30 µM), and PAR2-AP (30 µM) were inhibited by 1 µM of AZ3451, a potent and selective PAR2 antagonist.

Article Snippet: The PAR2 coding sequence was amplified by polymerase chain reaction (PCR) using PAR2 plasmid (Genbank Accession No. NM_005252.5) purchased from OriGene Technologies (Rockville, MD, USA) as the template (forward primer: TTTTT GAATTC CACC ATG CGG AGC CCC AGC, reverse primer: TTTTT TCTAGA TTA ATA GGA GGT CTT AAC AGT GG).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: GB83, an Agonist of PAR2 with a Unique Mechanism of Action Distinct from Trypsin and PAR2-AP

doi: 10.3390/ijms231810631

Figure Lengend Snippet: GB83 potently induced a PAR2-mediated increase in intracellular calcium concentration. ( A ) Effect of PAR2-AP (30 µM), GB83 (30 µM), and PAR1-AP (30 µM) on intracellular calcium levels in A2058 cells not expressing PAR2. ( B , C ) Effect of PAR2-AP and GB83 on PAR2-mediated intracellular calcium increase in A2058 cells stably transfected with human PAR2. Arrowheads indicate when PAR2-AP and GB83 were applied. AZ3451 (1 µM) was pretreated 15 min before application of PAR2-AP and GB83. ( D ) Dose–response curve for GB83-induced PAR2 activation (mean ± S.E., n = 5).

Article Snippet: The PAR2 coding sequence was amplified by polymerase chain reaction (PCR) using PAR2 plasmid (Genbank Accession No. NM_005252.5) purchased from OriGene Technologies (Rockville, MD, USA) as the template (forward primer: TTTTT GAATTC CACC ATG CGG AGC CCC AGC, reverse primer: TTTTT TCTAGA TTA ATA GGA GGT CTT AAC AGT GG).

Techniques: Concentration Assay, Expressing, Stable Transfection, Transfection, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: GB83, an Agonist of PAR2 with a Unique Mechanism of Action Distinct from Trypsin and PAR2-AP

doi: 10.3390/ijms231810631

Figure Lengend Snippet: GB83 induced phosphorylation of ERK1/2 through PAR2 activation in HT-29 cells. ( A ) Representative immunoblots of ERK1/2 phosphorylation. PAR2 stimulated by trypsin (30 U/mL), GB83 (30 µM), and PAR2-AP (30 µM). ( B ) p-ERK1/2 band intensity was normalized to β-actin (mean ± S.E., n = 5). Statistical significance of differences was assessed by one-way ANOVA with Tukey’s post-hoc test. * p < 0.05, Trypsin and PAR2-AP versus GB83. ( C ) PAR2-mediated phosphorylation of ERK1/2 by GB83. AZ3451 (1 µM) was pretreated 15 min prior to stimulation with GB83 (30 µM). ( D ) p-ERK1/2 band intensity was normalized to β-actin (mean ± S.E., n = 5). Statistical significance of differences between indicated groups was assessed by one-way ANOVA with Tukey’s post-hoc test. ** p < 0.01.

Article Snippet: The PAR2 coding sequence was amplified by polymerase chain reaction (PCR) using PAR2 plasmid (Genbank Accession No. NM_005252.5) purchased from OriGene Technologies (Rockville, MD, USA) as the template (forward primer: TTTTT GAATTC CACC ATG CGG AGC CCC AGC, reverse primer: TTTTT TCTAGA TTA ATA GGA GGT CTT AAC AGT GG).

Techniques: Activation Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: GB83, an Agonist of PAR2 with a Unique Mechanism of Action Distinct from Trypsin and PAR2-AP

doi: 10.3390/ijms231810631

Figure Lengend Snippet: GB83 induced phosphorylation of p38 MAPK through PAR2 activation in HT-29 cells. ( A ) Representative immunoblots of p38 phosphorylation. PAR2 stimulated by trypsin (30 U/mL), GB83 (30 µM), and PAR2-AP (30 µM). ( B ) p-p38 band intensity was normalized to β-actin (mean ± S.E., n = 5). Statistical significance of differences was assessed by one-way ANOVA with Tukey’s post-hoc test. * p < 0.05 and ** p < 0.01 compared to GB83. ( C ) PAR2-mediated phosphorylation of p38 by GB83. AZ3451 (1 µM) was pretreated 15 min prior to stimulation with GB83 (30 µM). ( D ) p-p38 band intensity was normalized to β-actin (mean ± S.E., n = 5). Statistical significance of differences between indicated groups was assessed by one-way ANOVA with Tukey’s post-hoc test. *** p < 0.001.

Article Snippet: The PAR2 coding sequence was amplified by polymerase chain reaction (PCR) using PAR2 plasmid (Genbank Accession No. NM_005252.5) purchased from OriGene Technologies (Rockville, MD, USA) as the template (forward primer: TTTTT GAATTC CACC ATG CGG AGC CCC AGC, reverse primer: TTTTT TCTAGA TTA ATA GGA GGT CTT AAC AGT GG).

Techniques: Activation Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: GB83, an Agonist of PAR2 with a Unique Mechanism of Action Distinct from Trypsin and PAR2-AP

doi: 10.3390/ijms231810631

Figure Lengend Snippet: GB83 selectively and strongly internalized PAR2 in HT-29 cells. ( A ) EGFP-tagged PAR2 expressing HT-29 cells were treated with trypsin (30 U/mL), GB83 (30 µM), PAR2-AP (30 µM), and PAR4-AP (100 µM). Changes in cellular localization of EGFP-tagged PAR2 were observed at the indicated time points. ( B ) Quantitative analysis of the effect of trypsin, GB83, PAR2-AP, and PAR4-AP on PAR2 internalization. Summary of fold change in the number of puncta (mean ± S.E., n = 5). ( C ) EGFP-tagged PAR4 expressing HT-29 cells were treated with trypsin (30 U/mL), GB83 (30 µM), PAR2-AP (30 µM), and PAR4-AP (100 µM). Changes in cellular localization of EGFP-tagged PAR4 were observed at the indicated time points. ( D ) Quantitative analysis of the effect of trypsin, GB83, PAR2-AP, and PAR4-AP on PAR2 and PAR4 internalization. Summary of fold change in the number of puncta at 60 min (mean ± S.E., n = 5). Statistical significance of differences between indicated group with that before treatment was assessed by one-way ANOVA with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The PAR2 coding sequence was amplified by polymerase chain reaction (PCR) using PAR2 plasmid (Genbank Accession No. NM_005252.5) purchased from OriGene Technologies (Rockville, MD, USA) as the template (forward primer: TTTTT GAATTC CACC ATG CGG AGC CCC AGC, reverse primer: TTTTT TCTAGA TTA ATA GGA GGT CTT AAC AGT GG).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: GB83, an Agonist of PAR2 with a Unique Mechanism of Action Distinct from Trypsin and PAR2-AP

doi: 10.3390/ijms231810631

Figure Lengend Snippet: GB83 induces sustained colocalization of PAR2 and β-arrestin1. ( A – C ) Effect of trypsin, GB83, and PAR2-AP on the localization of PAR2 and β-arrestin1 (βarr1) in HT-29 cells expressing EGFP-tagged PAR2 and RFP-tagged β-arrestin1. Cells were treated with trypsin (30 U/mL), GB83 (30 µM), PAR2-AP (30 µM), and the cellular localization of PAR2 (green) and β-arrestin1 (red) were observed at 0, 10, 30, and 60 min after treatment of each agonist. ( D ) Quantitative analysis of colocalization of PAR2 and β-arrestin1 (mean ± S.E., n = 5). Colocalization values were calculated in Pearson’s correlation coefficient using the colocalization plugin JACoP. Statistical significance of differences between indicated groups was assessed by one-way ANOVA with Tukey’s post-hoc test. *** p < 0.001, ns, not significant.

Article Snippet: The PAR2 coding sequence was amplified by polymerase chain reaction (PCR) using PAR2 plasmid (Genbank Accession No. NM_005252.5) purchased from OriGene Technologies (Rockville, MD, USA) as the template (forward primer: TTTTT GAATTC CACC ATG CGG AGC CCC AGC, reverse primer: TTTTT TCTAGA TTA ATA GGA GGT CTT AAC AGT GG).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: GB83, an Agonist of PAR2 with a Unique Mechanism of Action Distinct from Trypsin and PAR2-AP

doi: 10.3390/ijms231810631

Figure Lengend Snippet: GB83 induces sustained colocalization of PAR2 and β-arrestin2. ( A – C ) Effect of trypsin, GB83, and PAR2-AP on the localization of PAR2 and β-arrestin2 (βarr2) in HT-29 cells expressing EGFP-tagged PAR2 and RFP-tagged β-arrestin2. Cells were treated with trypsin (30 U/mL), GB83 (30 µM), PAR2-AP (30 µM), and the cellular localization of PAR2 (green) and β-arrestin2 (red) were observed at 0, 10, 30, and 60 min after treatment of each agonist. ( D ) Quantitative analysis of colocalization of PAR2 and β-arrestin2 (mean ± S.E., n = 5). Colocalization values were calculated in Pearson’s correlation coefficient using the colocalization plugin JACoP. Statistical significance of differences between indicated groups was assessed by one-way ANOVA with Tukey’s post-hoc test. ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The PAR2 coding sequence was amplified by polymerase chain reaction (PCR) using PAR2 plasmid (Genbank Accession No. NM_005252.5) purchased from OriGene Technologies (Rockville, MD, USA) as the template (forward primer: TTTTT GAATTC CACC ATG CGG AGC CCC AGC, reverse primer: TTTTT TCTAGA TTA ATA GGA GGT CTT AAC AGT GG).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: GB83, an Agonist of PAR2 with a Unique Mechanism of Action Distinct from Trypsin and PAR2-AP

doi: 10.3390/ijms231810631

Figure Lengend Snippet: GB83 induced prolonged PAR2 desensitization and slow recovery of PAR2 in HT-29 cells. ( A ) Effect of trypsin, GB83, and PAR2-AP on cellular localization of EGFP-tagged PAR2 were observed at the indicated time points. Cells were treated with trypsin (30 U/mL), GB83 (30 μM) or PAR2-AP (30 μM) for 30 min, washed 3 times with PBS, and then replaced with culture medium. ( B – D ) Effect of trypsin, GB83, and PAR2-AP on functional recovery of PAR2. HT-29 cells were treated with trypsin (30 U/mL), GB83 (30 µM), or PAR2-AP (30 µM) for 30 min, washed 3 times with PBS, and then replaced with culture medium. PAR2-AP-mediated increases in intracellular calcium levels were measured at the indicated time points after wash out. ( E ) Summary of functional recovery of PAR2 (mean ± S.E., n = 5). Statistical significance of differences was assessed by one-way ANOVA with Tukey’s post-hoc test. ** p < 0.01 and *** p < 0.001 compared to GB83; ### p < 0.001 compared to trypsin.

Article Snippet: The PAR2 coding sequence was amplified by polymerase chain reaction (PCR) using PAR2 plasmid (Genbank Accession No. NM_005252.5) purchased from OriGene Technologies (Rockville, MD, USA) as the template (forward primer: TTTTT GAATTC CACC ATG CGG AGC CCC AGC, reverse primer: TTTTT TCTAGA TTA ATA GGA GGT CTT AAC AGT GG).

Techniques: Functional Assay

Journal: Acta pharmacologica Sinica

Article Title: Epigallocatechin-3-gallate inhibits proliferation and migration of human colon cancer SW620 cells in vitro.

doi: 10.1038/aps.2011.139

Figure Lengend Snippet: Figure 1. Effects of EGCG on PAR2-AP- and factor VIIa-induced proliferation and migration of SW620 cells. (A) SW620 cells were plated at 5×104 cells in 96-well plates and pretreated with different concentrations of EGCG (0, 25, 50, 75, and 100 μg/mL) for 15 min, then incubated with PAR2-AP (100 μmol/L) or factor VIIa (10 nmol/L) for 24 h. The above EGCG was kept in media. The cell proliferation was measured using MTT colorimetric assay. n=6. Mean±SEM. eP<0.05, fP<0.01 vs PAR2-AP- or factor VIIa-stimulated alone. (B) The cells were pretreated with or without EGCG (100 μg/mL) for 15 min as described above and then incubated with PAR2-AP (100 μmol/L) or factor VIIa (10 nmol/L) for 8 h. The cell migratory potential was measured by modified Boyden Chambers as described in Methods. n=3. Mean±SEM. aP>0.05, cP<0.01 vs media only; eP<0.05, fP<0.01 vs PAR2-AP- or factor VIIa-stimulated alone.

Article Snippet: The PAR2 agonist (SLIGKV-NH2, PAR2-AP) was synthesized by Proteintech Group Inc (Wuhan, China).

Techniques: Migration, Incubation, Colorimetric Assay, Modification

Journal: Acta pharmacologica Sinica

Article Title: Epigallocatechin-3-gallate inhibits proliferation and migration of human colon cancer SW620 cells in vitro.

doi: 10.1038/aps.2011.139

Figure Lengend Snippet: Figure 2. Fluorescence photomicrographs of SW620 cells with rhodamin-conjugated phalloidin and Hoechst 33342 staining (×400). SW620 cells were placed in 24-well culture dishes at 3×104 cells/well and treated with different conditions for 1 h. Then, the cells were fixed with 4% paraformaldehyde and washed with PBS three times. The DNA was stained with Hoechst 33342 (blue) and the actin cytoskeleton with rhodamin- conjugated phalloidin (red) which were observed under a laser scanning confocal fluorescence microscope. (A) Media only; (B) With 100 μmol/L of PAR2-AP; (C) With 10 nmol/L of factor VIIa; (D) With 100 μg/mL of EGCG; (E) With EGCG (100 μg/mL)/ PAR2-AP (100 μmol/L); (F) With EGCG (100 μg/mL)/factor VIIa (10 nmol/L).

Article Snippet: The PAR2 agonist (SLIGKV-NH2, PAR2-AP) was synthesized by Proteintech Group Inc (Wuhan, China).

Techniques: Fluorescence, Staining, Microscopy

Journal: Acta pharmacologica Sinica

Article Title: Epigallocatechin-3-gallate inhibits proliferation and migration of human colon cancer SW620 cells in vitro.

doi: 10.1038/aps.2011.139

Figure Lengend Snippet: Figure 3. Effects of EGCG on caspase-7 expression in SW620 cells. SW620 cells (1×106–1×107) were stimulated with PAR2-AP (100 μmol/L) or factor VIIa (10 nmol/L) in the absence or presence of EGCG (100 μg/ mL) for 2 h or 24 h. Then the total RNAs (2 h) and cell lysates (24 h) were collected for caspase-7 mRNA (A) and its protein (B) determination using QT-PCR and Western blot analysis, respectively. The caspase-7 mRNA levels were normalized to control values of β-actin and its protein levels were expressed as the ratio of caspase-7/β-actin bands density. n=3. Mean±SEM. aP>0.05, cP<0.01 vs media only; fP<0.01 vs PAR2-AP- or factor VIIa-stimulated alone.

Article Snippet: The PAR2 agonist (SLIGKV-NH2, PAR2-AP) was synthesized by Proteintech Group Inc (Wuhan, China).

Techniques: Expressing, Western Blot, Control

Journal: Acta pharmacologica Sinica

Article Title: Epigallocatechin-3-gallate inhibits proliferation and migration of human colon cancer SW620 cells in vitro.

doi: 10.1038/aps.2011.139

Figure Lengend Snippet: Figure 4. Effect of EGCG on the expression of TF and MMP-9 in SW620 cells. The cells (1×106) were incubated with PAR2-AP (100 μmol/L) or factor VIIa (10 nmol/L) in the absence or presence of EGCG (100 μg/L) for 24 h. The cell lysates were prepared and the TF activity (A) as well as MMP-9 levels (B) was measured by the specific kits, respectively. n=3. Mean±SEM. aP>0.05, bP<0.05, cP<0.01 vs media only; fP<0.01 vs PAR2- AP- or factor VIIa-stimulated alone.

Article Snippet: The PAR2 agonist (SLIGKV-NH2, PAR2-AP) was synthesized by Proteintech Group Inc (Wuhan, China).

Techniques: Expressing, Incubation, Activity Assay

Journal: Acta pharmacologica Sinica

Article Title: Epigallocatechin-3-gallate inhibits proliferation and migration of human colon cancer SW620 cells in vitro.

doi: 10.1038/aps.2011.139

Figure Lengend Snippet: Figure 5. Effects of EGCG on activation of ERK1/2 and NF-κB pathway in SW620 cells. The cells were pretreated with or without the indicated concentrations of EGCG for 15 min and then incubated with PAR2-AP (100 μmol/L) or factor VIIa (10 nmol/L) for indicated time. The above EGCG was kept in media. The cytoplasmic and nuclear lysates of the cells were collected and subjected to Western analysis with antibodies to total and phosphorylated ERK1/2 (A) and to NF-κB/p65 as well as Histon H3 (B). n=3. Mean±SEM. aP>0.05, bP<0.05, cP<0.01 vs control; dP>0.05,

Article Snippet: The PAR2 agonist (SLIGKV-NH2, PAR2-AP) was synthesized by Proteintech Group Inc (Wuhan, China).

Techniques: Activation Assay, Incubation, Western Blot, Control