Journal: Brazilian Oral Research

Article Title: Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation

doi: 10.1590/1807-3107bor-2024.vol38.0079

Figure Lengend Snippet: Cell sheet culture and osteogenic differentiation. Representative images. A) a) Macroscopic aspect of cell sheet detaching from the culture plate; b) Microscopic aspect of a detail (scale bar = 100 μm); c) Macroscopic aspect of detached cell sheet; d) Microscopic aspect of a detail (scale bar = 100 μm). B) Microscopic aspect of cultured cell sheets after 14 days of induction of osteogenic differentiation with a) Clonogenic (control), b) Osteogenic, and c) Osteogenic+PAR1 medium (scale bar = 100 μm). C) Cell proliferation (WST-1 Assay). D) Gene expression during induction of osteogenic differentiation in vitro (β-GAL). Calibrating sample: undifferentiated PDLSCs (P4). Housekeeping gene: GAPDH. E) Quantification of alkaline phosphatase (ALP) activity. F) Macroscopic aspect of mineralized nodules formed after 2, 7, and 14 days of induction of osteogenic differentiation by Alizarin Red S staining in the analyzed groups. G) Quantification of Alizarin Red S Assay.

Article Snippet: Amplicons were generated using the following probes: Alkaline Phosphatase (ALP, Hs03046558_s1), β-galactosidase (GLB1, Hs01035168_m1), Collagen I (COL1A1, Hs00164004_m1), Osteocalcin (OC, Hs00609452_g1), Osteoprotegerin (OPG, Hs00171068_m1), Osterix (Sp7, Hs01866874_s1), PAR 1 (Hs00169258_m1), Periostin (POSTN, Hs01566734_m1), Receptor activator of nuclear factor kappa B ligand (RANKL, Hs00243519_m1), Runt-related transcription factor 2 (RUNX2, Hs00231692_m1), β-actin (ACTB, Hs99999903_m1), and GAPDH (Hs02786624_g1) (Applied Biosystems, Foster City, CA, USA).

Techniques: Cell Culture, Control, WST-1 Assay, Gene Expression, In Vitro, Activity Assay, Staining

Journal: Brazilian Oral Research

Article Title: Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation

doi: 10.1590/1807-3107bor-2024.vol38.0079

Figure Lengend Snippet: A) to I) Gene expression during induction of osteogenic differentiation in vitro of PDLSCs and cell sheet (RUNX2, SP7, OPG, RANKL, PAR1, POSTN, OC, COL I, and ALP) for 2, 7, and 14 days. Calibrating sample: undifferentiated PDLSCs (P4). Housekeeping gene: GAPDH. Gray bar indicates osteogenic and black bar indicates osteogenic + PAR1 medium group.

Article Snippet: Amplicons were generated using the following probes: Alkaline Phosphatase (ALP, Hs03046558_s1), β-galactosidase (GLB1, Hs01035168_m1), Collagen I (COL1A1, Hs00164004_m1), Osteocalcin (OC, Hs00609452_g1), Osteoprotegerin (OPG, Hs00171068_m1), Osterix (Sp7, Hs01866874_s1), PAR 1 (Hs00169258_m1), Periostin (POSTN, Hs01566734_m1), Receptor activator of nuclear factor kappa B ligand (RANKL, Hs00243519_m1), Runt-related transcription factor 2 (RUNX2, Hs00231692_m1), β-actin (ACTB, Hs99999903_m1), and GAPDH (Hs02786624_g1) (Applied Biosystems, Foster City, CA, USA).

Techniques: Gene Expression, In Vitro

Journal: Brazilian Oral Research

Article Title: Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation

doi: 10.1590/1807-3107bor-2024.vol38.0079

Figure Lengend Snippet: A) to F) Protein levels (ELISA) during induction of osteogenic differentiation in vitro of PDLSCs and cell sheet (OPG, RANKL, PAR1, POSTN, and OC) for 2, 7, and 14 days.

Article Snippet: Amplicons were generated using the following probes: Alkaline Phosphatase (ALP, Hs03046558_s1), β-galactosidase (GLB1, Hs01035168_m1), Collagen I (COL1A1, Hs00164004_m1), Osteocalcin (OC, Hs00609452_g1), Osteoprotegerin (OPG, Hs00171068_m1), Osterix (Sp7, Hs01866874_s1), PAR 1 (Hs00169258_m1), Periostin (POSTN, Hs01566734_m1), Receptor activator of nuclear factor kappa B ligand (RANKL, Hs00243519_m1), Runt-related transcription factor 2 (RUNX2, Hs00231692_m1), β-actin (ACTB, Hs99999903_m1), and GAPDH (Hs02786624_g1) (Applied Biosystems, Foster City, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Brazilian Oral Research

Article Title: Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation

doi: 10.1590/1807-3107bor-2024.vol38.0079

Figure Lengend Snippet: Cell sheet technique. Immediately after extraction, human teeth were transported to the laboratory in a tube with culture medium and antibiotics. The periodontal ligament (PDL) was scaled, fragmented, and cultured in flasks. PDLSCs were isolated and cultured with a specific medium to obtain the cell sheets. PAR1 activation increased osteogenic activity and decreased senescence in cell sheets.

Article Snippet: Amplicons were generated using the following probes: Alkaline Phosphatase (ALP, Hs03046558_s1), β-galactosidase (GLB1, Hs01035168_m1), Collagen I (COL1A1, Hs00164004_m1), Osteocalcin (OC, Hs00609452_g1), Osteoprotegerin (OPG, Hs00171068_m1), Osterix (Sp7, Hs01866874_s1), PAR 1 (Hs00169258_m1), Periostin (POSTN, Hs01566734_m1), Receptor activator of nuclear factor kappa B ligand (RANKL, Hs00243519_m1), Runt-related transcription factor 2 (RUNX2, Hs00231692_m1), β-actin (ACTB, Hs99999903_m1), and GAPDH (Hs02786624_g1) (Applied Biosystems, Foster City, CA, USA).

Techniques: Extraction, Cell Culture, Isolation, Activation Assay, Activity Assay

Journal: Brazilian Oral Research

Article Title: Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation

doi: 10.1590/1807-3107bor-2024.vol38.0079

Figure Lengend Snippet: A) to H) Gene expression during induction of osteogenic in vitro differentiation and inhibition of signaling pathways (Wnt/β-catenin, TGF-βRI, MEK, p38 MAPK, and FGFR/VEGFR) of PDLSCs (RUNX2, SP7, OPG, RANKL, PAR1, POSTN, OC, and COL I) for 2, 7, and 14 days. Calibrating sample: undifferentiated PDLSCs (P4). Housekeeping gene: GAPDH.

Article Snippet: Amplicons were generated using the following probes: Alkaline Phosphatase (ALP, Hs03046558_s1), β-galactosidase (GLB1, Hs01035168_m1), Collagen I (COL1A1, Hs00164004_m1), Osteocalcin (OC, Hs00609452_g1), Osteoprotegerin (OPG, Hs00171068_m1), Osterix (Sp7, Hs01866874_s1), PAR 1 (Hs00169258_m1), Periostin (POSTN, Hs01566734_m1), Receptor activator of nuclear factor kappa B ligand (RANKL, Hs00243519_m1), Runt-related transcription factor 2 (RUNX2, Hs00231692_m1), β-actin (ACTB, Hs99999903_m1), and GAPDH (Hs02786624_g1) (Applied Biosystems, Foster City, CA, USA).

Techniques: Gene Expression, In Vitro, Inhibition, Protein-Protein interactions

Journal: Brazilian Oral Research

Article Title: Cell sheet produced from periodontal ligament stem cells activated by PAR1 improves osteogenic differentiation

doi: 10.1590/1807-3107bor-2024.vol38.0079

Figure Lengend Snippet: A) Gene expression during induction of in vitro osteogenic differentiation and inhibition of signaling pathways (Wnt/β-catenin, TGF-βRI, MEK, p38 MAPK, and FGFR/VEGFR) of PDLSCs (ALP) for 2, 7, and 14 days. Calibrating sample: undifferentiated PDLSCs (P4). Housekeeping gene: GAPDH. B) to G) Protein levels (ELISA) during induction of in vitro osteogenic differentiation and inhibition of signaling pathways (Wnt/β-catenin, TGF-βRI, MEK, p38 MAPK, and FGFR/VEGFR) of PDLSCs (OPG, RANKL, PAR1, POSTN, OC, and COL I) for 2, 7 and 14 days.

Article Snippet: Amplicons were generated using the following probes: Alkaline Phosphatase (ALP, Hs03046558_s1), β-galactosidase (GLB1, Hs01035168_m1), Collagen I (COL1A1, Hs00164004_m1), Osteocalcin (OC, Hs00609452_g1), Osteoprotegerin (OPG, Hs00171068_m1), Osterix (Sp7, Hs01866874_s1), PAR 1 (Hs00169258_m1), Periostin (POSTN, Hs01566734_m1), Receptor activator of nuclear factor kappa B ligand (RANKL, Hs00243519_m1), Runt-related transcription factor 2 (RUNX2, Hs00231692_m1), β-actin (ACTB, Hs99999903_m1), and GAPDH (Hs02786624_g1) (Applied Biosystems, Foster City, CA, USA).

Techniques: Gene Expression, In Vitro, Inhibition, Protein-Protein interactions, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression

doi: 10.3389/fmolb.2023.1211597

Figure Lengend Snippet: APC-mediated attenuation of TNF-α-induced VCAM-1 expression requires PAR1 and S1PR1. (A) Endothelial cells were pre-incubated with Vorapaxar (PAR1 antagonist), W146 (S1PR1 antagonist) or DMSO vehicle control, prior to pretreatment with APC for 30 min and then treated with TNF-α. Cell lysates were immunoblotted for VCAM-1 and GAPDH. Quantification of VCAM-1 induction (mean ± S.D.) from three independent experiments were analyzed by one-way ANOVA; ****, p < 0.0001; n. s., not significant. (B,C) Endothelial cells were transfected with non-specific (NS), PAR1, or PAR3 specific siRNA. The relative PAR1 (B) and PAR3 (C) mRNA expression levels were quantified by RT-qPCR. Results were normalized to 18S mRNA expression and are represented as fold-change relative to non-specific siRNA control (mean ± S.D.) from three independent experiments and analyzed one-way ANOVA; **, p < 0.01; ****, p < 0.0001. (D) Endothelial cells were transfected with NS, PAR1, or PAR3 specific siRNA, pretreated with or without APC and then stimulated with TNF-α for 24 h. Cell lysates were immunoblotted for VCAM-1 and GAPDH expression. Quantification of VCAM-1 induction (mean ± S.D.) from three independent experiments was examined by one-way ANOVA; ****, p < 0.0001; n. s., not significant.

Article Snippet: Quantitative RT-PCR was performed with TaqMan Fast Advanced Master Mix (#4444964, ThermoFisher Scientific) and TaqMan Gene Expression Probes including: PAR1 ( F2R ) (ThermoFisher, #Hs05045041_s1), PAR3 (F2RL3 ) (ThermoFisher, #Hs00187982_m1), VCAM-1 ( VCAM1 ) (ThermoFisher, #Hs01003372_m1) and TNF-α (ThermoFisher, #Hs00174128_m1).

Techniques: Expressing, Incubation, Transfection, Quantitative RT-PCR

Journal: Frontiers in Molecular Biosciences

Article Title: Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression

doi: 10.3389/fmolb.2023.1211597

Figure Lengend Snippet: APC-mediated attenuation of TNF-α-induced VCAM-1 expression requires GRK2. (A) Endothelial cells transfected with non-specific (NS), GRK2, or GRK5 siRNA were pretreated with or without APC and then stimulated with TNF-α. Cell lysates were immunoblotted for VCAM-1, GRK2/3, GRK4/5/6 and GAPDH expression. Quantification of VCAM-1 expression (mean ± S.D.) from three independent experiments were analyzed by one-way ANOVA; ***, p < 0.001; n. s., not significant. (B) Endothelial cells preincubated with Vorapaxar (PAR1 antagonist) or CMPD101 (GRK2 inhibitor) were pretreated with APC and then stimulated with TNF-α. Cell lysates were immunoblotted for VCAM-1 and GAPDH. Quantification of VCAM-1 expression (mean ± S.D.) from three independent experiments were analyzed by one-way ANOVA; ***, p < 0.001; n. s., not significant. (C) HEK293 cells transfected with PAR1-YFP and β-arr2-Rluc coexpressing EPCR were pretreated with CMPD101 or DMSO vehicle control, stimulated with APC and β-arr2 recruitment to PAR1 was determined by BRET. Data (mean ± S.D.) from three independent experiments were analyzed by one-way ANOVA; ***, p < 0.001.

Article Snippet: Quantitative RT-PCR was performed with TaqMan Fast Advanced Master Mix (#4444964, ThermoFisher Scientific) and TaqMan Gene Expression Probes including: PAR1 ( F2R ) (ThermoFisher, #Hs05045041_s1), PAR3 (F2RL3 ) (ThermoFisher, #Hs00187982_m1), VCAM-1 ( VCAM1 ) (ThermoFisher, #Hs01003372_m1) and TNF-α (ThermoFisher, #Hs00174128_m1).

Techniques: Expressing, Transfection

Journal: Frontiers in Molecular Biosciences

Article Title: Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression

doi: 10.3389/fmolb.2023.1211597

Figure Lengend Snippet: Model of APC/PAR1-S1PR1 mediated protection against TNF-α induced VCAM1 expression. In endothelial cells, APC bound to EPCR cleaves and activates PAR1 to stimulate β-arr2 mediated cytoprotection against TNF-α induced upregulation of adhesion molecule VCAM-1 expression. Interestingly, APC/PAR1-mediated cytoprotection against TNF-α-induced VCAM-1 expression further requires the co-receptor S1PR1 and unexpectedly is dependent on GRK2. These studies highlight the complexity of APC/PAR1 cytoprotective signaling, which utilizes multiple co-receptors and different GRKs to promote distinct β-arr2-dependent cytoprotective responses in endothelial cells. The mechanism by which APC/PAR1 signaling intersects with the TNF-α-stimulated AP-1, p38 MAPK and NF-κB pathways to promote gene transcription of VCAM-1 expression is not known.

Article Snippet: Quantitative RT-PCR was performed with TaqMan Fast Advanced Master Mix (#4444964, ThermoFisher Scientific) and TaqMan Gene Expression Probes including: PAR1 ( F2R ) (ThermoFisher, #Hs05045041_s1), PAR3 (F2RL3 ) (ThermoFisher, #Hs00187982_m1), VCAM-1 ( VCAM1 ) (ThermoFisher, #Hs01003372_m1) and TNF-α (ThermoFisher, #Hs00174128_m1).

Techniques: Expressing

Journal: Frontiers in Genetics

Article Title: Whole Transcriptome Analysis Identifies the Taxonomic Status of a New Chinese Native Cattle Breed and Reveals Genes Related to Body Size

doi: 10.3389/fgene.2020.562855

Figure Lengend Snippet: 51 genes with P -values of less than 0.05 according to four independent gene-level LOF variant enrichment analysis methods.

Article Snippet: Additionally, three genes with a suggestive association were found: ESPNL (espin-like), PRCP (prolylcarboxypeptidase) and F2R (coagulation factor II thrombin receptor) (P_Chi < 1e-4).

Techniques: Variant Assay

Journal: Science Advances

Article Title: A CRISPR activation screen reveals a cilia disassembly pathway mutated in focal cortical dysplasia

doi: 10.1126/sciadv.aeb7238

Figure Lengend Snippet: ( A ) Illustration of F2R highlighting its activation, inhibition by vorapaxar, and signaling through G proteins. ( B ) Parental NIH-3T3 cells and cells transduced with F2r CRISPRa sgRNAs were serum starved for 24 hours, followed by immunofluorescence analysis of cilia (Arl13b) and centrioles [γ-tubulin (γ-tub)]. Where indicated, cells were treated with 6 μM vorapaxar during serum starvation. ( C ) Quantification of ciliation from (B). Bars show means of N = 3 independent replicates (N > 75 cells each). ( D ) Diagram of SARM1, including NADase TIR domain that cleaves NAD + to produce nicotinamide (NAM) and cADPR. ( E ) Analysis of ciliation in NIH-3T3 cell lines as in (A). Where indicated, cells were treated with 30 μM DSRM-3716 during serum starvation. ( F ) Quantification of ciliation from (E). Bars show means of N = 3 independent replicates ( N > 80 cells each). ( G ) Analysis of ciliation in NIH-3T3 cells expressing doxycycline (Dox)–inducible F2r (Tet-F2r-Flag). Cells were treated with Dox (1 μg/ml) and vorapaxar during serum starvation. Bars show means of N = 3 independent experiments ( N > 125 cells each). ( H ) Analysis of ciliation in NIH-3T3 Tet-F2r-Flag cells treated as indicated. Lines show means from N = 3 independent experiments ( N > 95 cells each). ( I ) Analysis of ciliation in serum-starved RPE1 cells after treatment with thrombin (10 U/ml). Thin lines show N = 3 replicates ( N > 100 cells each); thick lines show means. ( J ) Live-cell imaging of RPE1 cells with labeled cilia (Htr6-3xmNeonGreen) and centrioles (miRFP670-Centrin2). Time after thrombin addition is noted in hour:minute:second. See also fig. S3C. Scale bars, 5 μm. Error bars show standard deviations; asterisks denote significant (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001) differences in mean, as determined by two-sided t test.

Article Snippet: PB-Tet-F2r-3xFlag was cloned by Gibson assembly of a synthesized mouse F2r gene product (Twist Biosciences) into a piggyBac Tet-Cas9 plasmid with Neo resistance provided by I. Cheeseman (Massachusetts Institute of Technology).

Techniques: Activation Assay, Inhibition, Transduction, Immunofluorescence, Expressing, Live Cell Imaging, Labeling

Journal: Science Advances

Article Title: A CRISPR activation screen reveals a cilia disassembly pathway mutated in focal cortical dysplasia

doi: 10.1126/sciadv.aeb7238

Figure Lengend Snippet: ( A ) Diagram of proposed cilia disassembly pathway. ( B ) Immunofluorescence analysis of ciliation in NIH-3T3 Tet-F2r-Flag cells treated with Dox (1 μg/ml), 30 μM DSRM-3716, and/or 10 μM dHNN during 24-hour serum starvation. Bars show means of N ≥ 3 independent experiments ( N > 100 cells each). ( C ) Analysis of ciliation in RPE1 cells. Cells were serum starved prior to addition of thrombin (10 U/ml), with or without vorapaxar or DSRM-3716. Bars show means of N = 3 independent experiments ( N > 140 cells each). ( D ) NIH-3T3 Tet-F2r-Flag cells were serum starved for 24 hours, followed by analysis of cilia (Arl13b) and centrioles (γ-tubulin). Cells were treated with Dox, 5 μM dantrolene, 5 μM BAPTA-AM, and/or 10 μM Y-27632 during serum starvation (dantrolene was added 24 hours prior to serum starvation). ( E ) Quantification of ciliation in NIH-3T3 Tet-F2r-Flag cells treated as in (D). Bars show means of N ≥ 3 independent experiments ( N > 85 cells each). ( F ) Left: Illustration of centrosome-targeted calcium sink based on PACT targeting domain and Pvalb calcium-binding domain. Right: Analysis of cilia, centrioles [marked by pericentrin (PCNT)], and PACT-Pvalb-mCherry in RPE1 cells following serum starvation and 24 hours thrombin treatment. Where indicated, cells were treated with Dox (20 ng/ml) during thrombin treatment and for 24 hours prior. ( G ) Quantification of ciliation in RPE1 cell lines with inducible transgenes for a centrosome-targeted calcium sink (PACT-Pvalb-mCherry), a nontargeted calcium sink (Pvalb-mCherry), or centrosome-targeted mCherry (PACT-mCherry). Bars show means of N ≥ 3 independent experiments ( N > 100 cells each). Scale bars, 5 μm. Error bars show standard deviations; asterisks denote significant (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., not significant) differences in mean, as determined by two-sided t test.

Article Snippet: PB-Tet-F2r-3xFlag was cloned by Gibson assembly of a synthesized mouse F2r gene product (Twist Biosciences) into a piggyBac Tet-Cas9 plasmid with Neo resistance provided by I. Cheeseman (Massachusetts Institute of Technology).

Techniques: Immunofluorescence, Binding Assay