Journal: Heliyon

Article Title: Long non-coding RNA AC245100.4 activates the PI3K/AKT pathway to promote PCa cell proliferation by elevating PAR2

doi: 10.1016/j.heliyon.2023.e16870

Figure Lengend Snippet: PAR2 is positively regulated by AC245100.4 and highly expressed in prostate cancer tissues and cells. (A) PAR2 protein expression in PCa cells with si- AC245100.4 or OE- AC245100.4 was tested via Western blot. (B)The expression of PAR2 in 24 tumor and 24 tumor and their adjacent tissues was from UALCN. (C) The expression of PAR2 in prostate cancer tissue samples (492) and prostate tissue samples (152) was from GEPIA2. (D) and (E) The expression of PAR2 in prostate cancer tissue samples and prostate tissue samples was from GSE46602 or GSE71016. (F) and (G) qRT-PCR and Western blot were used to detect PAR2 mRNA and protein expression in PC3, PC3M, DU145 and RWPE-1 cell lines. *P<0.05, **P<0.01, ***P<0.001 , n = 3 for each group.

Article Snippet: PAR2 (Proteintech, USA), p-AKT (Cell Signaling, USA), AKT (Cell Signaling, USA), Cyclin D1 (Biyuntian, China), and PCNA were the primary antibodies used for Western blot (Biyuntian, China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Heliyon

Article Title: Long non-coding RNA AC245100.4 activates the PI3K/AKT pathway to promote PCa cell proliferation by elevating PAR2

doi: 10.1016/j.heliyon.2023.e16870

Figure Lengend Snippet: AC245100.4 promotes cell proliferation via regulating PAR2 in PCa cells. (A) PAR2 protein expression in PCa cells with si- PAR2 or OE- PAR2 was tested via Western blot. (B) and (C) The cell proliferation was tested by CCK8 assay and clone formation assay in PCa cells with si- PAR2 or OE- PAR2 . (D) Western blot was used to detect Cyclin D1, PCNA protein expression in PCa cells with si- PAR2 or OE- PAR2 . (E) and (F) The cell proliferation was tested by CCK8 assay and clone formation assay in PCa cells with OE- AC245100.4 and si- PAR2 . *P<0.05, **P<0.01, ***P<0.001 , n = 3 for each group.

Article Snippet: PAR2 (Proteintech, USA), p-AKT (Cell Signaling, USA), AKT (Cell Signaling, USA), Cyclin D1 (Biyuntian, China), and PCNA were the primary antibodies used for Western blot (Biyuntian, China).

Techniques: Expressing, Western Blot, CCK-8 Assay, Tube Formation Assay

Journal: Heliyon

Article Title: Long non-coding RNA AC245100.4 activates the PI3K/AKT pathway to promote PCa cell proliferation by elevating PAR2

doi: 10.1016/j.heliyon.2023.e16870

Figure Lengend Snippet: AC245100.4 promotes cell proliferation via PAR2/PI3K/AKT axis in prostate cancer cells. (A) The distribution of ZSTK474 IC 50 score of each group of samples was predicated. (B)The correlation between PAR2 expression and PI3K/AKT/mTOR pathway scores was calculated. (C) AKT, p-AKT protein expression in PCa cells with si- AC245100.4 or OE- AC245100.4 were tested via Western blot. (D) AKT, p-AKT protein expression in PCa cells with si- PAR2 or OE- PAR2 was tested via Western blot. (E) ZSTK474 was added in PC3 cells with AC245100.4 overexpression，and the AKT, p-AKT protein expression were detected by Western Blot. (F) and (G) ZSTK474 was added in PC3 cells with AC245100.4 overexpression，and the proliferation levels in prostate cancer cells were measured using the CCK8 assay and clone formation assay. (H) AKT, p-AKT protein expression were tested in PCa cells with OE- AC245100.4 and si- PAR2 via Western blot. *P<0.05, **P<0.01, ***P<0.001 , n = 3 for each group.

Article Snippet: PAR2 (Proteintech, USA), p-AKT (Cell Signaling, USA), AKT (Cell Signaling, USA), Cyclin D1 (Biyuntian, China), and PCNA were the primary antibodies used for Western blot (Biyuntian, China).

Techniques: Expressing, Western Blot, Over Expression, CCK-8 Assay, Tube Formation 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: 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