Journal: Cellular and Molecular Neurobiology

Article Title: Procoagulant Extracellular Vesicles Increase Neuronal Tau expression, Metabolism and Processing Through Tissue Factor and Protease Activated Receptor 2

doi: 10.1007/s10571-025-01658-7

Figure Lengend Snippet: Examination of the mechanism of influence of TF on Tau protein expression and phosphorylation in differentiated SH-SY5Y cells. SH-SY5Y (2 × 10 5 ) were treated with recombinant relipidated Innovin TF (0.65 ng/ml) together with or without human fVIIa (5 nM) or fVIIa alone. In some experiments, the TF aliquots were pre-incubated for 1 h, with 10H10 antibody (20 µg/ml) capable of blocking TF signalling, HTF-1 antibody (20 µg/ml) to block TF-fVIIa protease/procoagulant activity, or a mouse control isotype IgG antibody (20 µg/ml; not shown). In other experiments, fVIIa was pre-incubated for 1 h with the chemical inhibitor PCI27483 (10 µg/ml). Alternatively, the neuronal cells were treated with AIIB2 antibody (20 µg/ml) to block β1-integrin signalling, SAM11 antibody (20 µg/ml) capable of blocking PAR2 signalling, or PAR2-activating peptide (PAR2-AP; 20 µM) to induce PAR2 signalling. Cells were harvested at 24 h, and cellular lysates (10 µg protein) were examined for Tau and phospho-Thr181 Tau by western blot analysis. All values were normalised against the respective GAPDH and for comparison, all ratios were calculated against the average from the non-treated cells ± the calculated standard deviation. The number of experiments is shown in each column, and all data groups were determined to have normal distributions and are shown in each column. A SH-SY5Y protein electrophoresis, B SH-SY5Y phospho-Thr181 Tau electrophoresis, C SH-SY5Y Tau protein ratio, and D SH-SY5Y phospho-Thr181 Tau ratio

Article Snippet: Alternatively, the neuronal cells were treated with a rat anti-human antibody (20 μg/ml; AIIB2; Merck KGaA) to block β1-integrin signalling, a mouse anti-human PAR2 antibody, SAM11 (20 μg/ml; Santa Cruz Biotechnology, Heidelberg, Germany), capable of blocking PAR2 signalling, or PAR2-activating peptide (PAR2-AP; 20 μM) to induce PAR2 signalling.

Techniques: Expressing, Phospho-proteomics, Recombinant, Incubation, Blocking Assay, Activity Assay, Control, Western Blot, Comparison, Standard Deviation, Protein Electrophoresis, Electrophoresis

Journal: Cellular and Molecular Neurobiology

Article Title: Procoagulant Extracellular Vesicles Increase Neuronal Tau expression, Metabolism and Processing Through Tissue Factor and Protease Activated Receptor 2

doi: 10.1007/s10571-025-01658-7

Figure Lengend Snippet: Time-course analysis of the Tau protein fragments in differentiated SH-SY5Y cells, following treatment with TF . SH-SY5Y (2 × 10 5 ) were treated with as single dose of recombinant relipidated Innovin TF (0.65 ng/ml) together with human fVIIa (5 nM). In some experiments, the TF aliquots were pre-incubated for 1 h, with 10H10 antibody (20 µg/ml) capable of blocking TF signalling, HTF-1 antibody (20 µg/ml) to block TF-fVIIa protease/procoagulant activity, or a mouse control isotype IgG antibody (20 µg/ml; not shown). Alternatively, the neuronal cells were treated with AIIB2 antibody (20 µg/ml) to block β1-integrin signalling, or SAM11 antibody (20 µg/ml) capable of blocking PAR2 signalling. Sets of cells were harvested at A 48 h and D at 72 h and cellular lysates (10 µg protein) were examined for Tau by western blot analysis. All values were normalised against the respective GAPDH (see Supplementary Fig. 6 A and B) and for comparison, all ratios were calculated against the average from the non-treated cells ± the calculated standard deviation. The data were obtained from 6 biological experiments, and all data groups were determined to have normal distributions. A Electrophoresis at 48 h, B calculated ratios of 50 kDa bands at 48 h, C calculated ratios of 30–35 kDa bands at 48 h, D electrophoresis at 72 h, E calculated ratios of 50 kDa bands at 72 h, F calculated ratios of 40 kDa bands at 72 h, G calculated ratios of 30–35 kDa bands at 72 h

Article Snippet: Alternatively, the neuronal cells were treated with a rat anti-human antibody (20 μg/ml; AIIB2; Merck KGaA) to block β1-integrin signalling, a mouse anti-human PAR2 antibody, SAM11 (20 μg/ml; Santa Cruz Biotechnology, Heidelberg, Germany), capable of blocking PAR2 signalling, or PAR2-activating peptide (PAR2-AP; 20 μM) to induce PAR2 signalling.

Techniques: Recombinant, Incubation, Blocking Assay, Activity Assay, Control, Western Blot, Comparison, Standard Deviation, Electrophoresis

Journal: Frontiers in Oncology

Article Title: Significance of FXa and its receptor PAR2 for the growth of colon cancer cells in vitro and in vivo

doi: 10.3389/fonc.2025.1631350

Figure Lengend Snippet: In vitro effects of FXa and PAR2 activation on proliferation and migration of murine CC cell line MC38 and involvement of EGFR. Proliferation. (A) Selective activation of PAR2 using AP2 induces significant proliferation, with the strongest stimulus being 10% FCS in the medium. Also, longterm stimulation with FXa enhances proliferation of the MC38 cell line. (B) Comparative analyses using BrdU assay exhibit equivalent increased proliferation rates after FXa stimulation of the cells. Migration. (C) Directed and (D) undirected cell migration of murine CC cells are significantly increased by FXa stimulation and selective PAR2 activation. Mean ± SD of n independent experiments is presented for all data. (A) n =7-11, (B) n =3-5, (C) n =5-7, (D) n =3, One-way ANOVA, Dunnett posthoc test, p < 0.05 (*). EGFR. (E) The involvement of EGFR was analyzed by pre-incubation of the cells with Erlotinib, an EGFR inhibitor, at varying concentrations and partially FXa stimulation. EGFR inhibition significantly decreased migration of the cell line used. Mean ± SD of n independent experiments is presented for all data. n =3-6, One-way ANOVA, Dunnett posthoc test, p < 0.05 (*). (F) The half-maximal inhibitory concentration (IC 50 ) of the EGFR-inhibitor, Erlotinib, was determined as 161 nM utilizing a resazurin-/crystal violet assay, n =3-6, Dose-response curves and ED50 values were derived through non-linear regression modelling (log(inhibitor) vs. normalized response - Variable slope).

Article Snippet: After blocking [Li-cor, Cat. #927-60001], membranes were incubated overnight at 4°C under rotating conditions with the following primary antibodies: AKT (1:1000) [Cell Signaling, Cat. #4691], phospho-AKT (1:1000) [Cell Signaling, Cat. #4060], p38 (1:500) [Cell Signaling, Cat. #8690], phospho-p38 (1:500) [Cell Signaling, Cat. #4511], p44/42 (1:1000) [Cell Signaling, Cat. #4695], phospho-p44/42 (1:1000) [Cell Signaling, Cat. #4370], PAR2 (1:200) [Santa Cruz Biotechnologies, Cat. #sc-13504], PAR1 (1:200) [Santa Cruz Biotechnologies, Cat. #13503].

Techniques: In Vitro, Activation Assay, Migration, BrdU Staining, Incubation, Inhibition, Concentration Assay, Crystal Violet Assay, Derivative Assay

Journal: Frontiers in Oncology

Article Title: Significance of FXa and its receptor PAR2 for the growth of colon cancer cells in vitro and in vivo

doi: 10.3389/fonc.2025.1631350

Figure Lengend Snippet: P38 MAPK, PI3K and EGFR signaling in proliferation and migration of MC38 cell line. The effects on proliferation and migration of the murine MC38 cells were evaluated through the use of specific inhibitors. The inhibited protein is indicated in brackets. Inhibition of p38 MAPK significantly decreased proliferation and migration rates of MC38 cells. PI3K and EGFR inhibition also resulted in reduced migration rates. PAR2 expression was analyzed via Western Blotting (M) The expression was normalized to total protein or β-Actin per lane. Shown is the fold induction relative to control. 30 nM FXa induced PAR2 protein level. Mean ± SD of n independent experiments is presented for all data. N =3-6, One-way ANOVA, Dunnett posthoc test, p < 0.05 (*).

Article Snippet: After blocking [Li-cor, Cat. #927-60001], membranes were incubated overnight at 4°C under rotating conditions with the following primary antibodies: AKT (1:1000) [Cell Signaling, Cat. #4691], phospho-AKT (1:1000) [Cell Signaling, Cat. #4060], p38 (1:500) [Cell Signaling, Cat. #8690], phospho-p38 (1:500) [Cell Signaling, Cat. #4511], p44/42 (1:1000) [Cell Signaling, Cat. #4695], phospho-p44/42 (1:1000) [Cell Signaling, Cat. #4370], PAR2 (1:200) [Santa Cruz Biotechnologies, Cat. #sc-13504], PAR1 (1:200) [Santa Cruz Biotechnologies, Cat. #13503].

Techniques: Migration, Inhibition, Expressing, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: Significance of FXa and its receptor PAR2 for the growth of colon cancer cells in vitro and in vivo

doi: 10.3389/fonc.2025.1631350

Figure Lengend Snippet: Characteristic in vivo values. Tail bleeding assay. (A) The direct FXa inhibitor Apixaban prolonged bleeding time significantly. Time was measured in mice that received 5 mg/kg Apixaban, 50 mg/kg Apixaban, or vehicle only (control) in both WT (white) and PAR2-KO (pink) mice. Mean ± SD of n independent experiments is presented for all data. n =5-6, Two-way ANOVA, p < 0.05 (*). Survival. (B) Kaplan-Meier estimates for treated (narrow line) and untreated (wide line) WT (black) and PAR2-KO (pink) mice. The survival duration of PAR2-KO animals was found to be significantly longer than that of WT animals. Apixaban did not demonstrate a substantial impact on this survival difference. Tumor growth in vivo . Untreated (C) and treated (D) WT (black) and PAR2-KO (pink) animals measured for maximum tumor extension. Apixaban demonstrates no significant effect.

Article Snippet: After blocking [Li-cor, Cat. #927-60001], membranes were incubated overnight at 4°C under rotating conditions with the following primary antibodies: AKT (1:1000) [Cell Signaling, Cat. #4691], phospho-AKT (1:1000) [Cell Signaling, Cat. #4060], p38 (1:500) [Cell Signaling, Cat. #8690], phospho-p38 (1:500) [Cell Signaling, Cat. #4511], p44/42 (1:1000) [Cell Signaling, Cat. #4695], phospho-p44/42 (1:1000) [Cell Signaling, Cat. #4370], PAR2 (1:200) [Santa Cruz Biotechnologies, Cat. #sc-13504], PAR1 (1:200) [Santa Cruz Biotechnologies, Cat. #13503].

Techniques: In Vivo, Control

Journal: Frontiers in Oncology

Article Title: Significance of FXa and its receptor PAR2 for the growth of colon cancer cells in vitro and in vivo

doi: 10.3389/fonc.2025.1631350

Figure Lengend Snippet: Characteristic in vivo values. Staining of resected tumors. Representative images depicting histological features of MC38 tumors of WT (row 1-2) and PAR2-KO mice (row 3-4) are shown. Haematoxylin and eosin (H&E) stained MC38 tumor sections were used to create overviews of the tumors in 1.1 mm depth (I-IV), blood vessels in 535 µm depth (V-VIII), mitotic active sections (IX-XII), and apoptotic areas in 270 µm (XIII-XVI). Hematoxylin stains all basophilic structures, such as the cell nucleus, blue, while eosin stains all basic structures red. These include the cytoplasm and extracellular components. A Pikro-Sirius red staining was performed at a depth of 535 µm to stain collagen fibers at the periphery of the tumors (XVII-XX). Overview staining was photographed at 20x magnification, vessels at 40x magnification, mitotic areas at 200x magnification, apoptotic areas at 100x magnification. The Pikro-Sirius staining as overview staining was magnified 10x.

Article Snippet: After blocking [Li-cor, Cat. #927-60001], membranes were incubated overnight at 4°C under rotating conditions with the following primary antibodies: AKT (1:1000) [Cell Signaling, Cat. #4691], phospho-AKT (1:1000) [Cell Signaling, Cat. #4060], p38 (1:500) [Cell Signaling, Cat. #8690], phospho-p38 (1:500) [Cell Signaling, Cat. #4511], p44/42 (1:1000) [Cell Signaling, Cat. #4695], phospho-p44/42 (1:1000) [Cell Signaling, Cat. #4370], PAR2 (1:200) [Santa Cruz Biotechnologies, Cat. #sc-13504], PAR1 (1:200) [Santa Cruz Biotechnologies, Cat. #13503].

Techniques: In Vivo, Staining

Journal: Frontiers in Oncology

Article Title: Significance of FXa and its receptor PAR2 for the growth of colon cancer cells in vitro and in vivo

doi: 10.3389/fonc.2025.1631350

Figure Lengend Snippet: Key figures of the animal study. Trial days. (A) Shown is the number of days the animals were subjected to the study, with a maximum of 21 days. PAR2-KO (black) mice stayed significantly longer in the trial than WT (white) mice. This finding is applicable to all three treatment groups. The animals were administered either 5 mg/kg or 50 mg/kg body weight Apixaban plus vehicle or vehicle only (control). Tumor size. (B) Additionally, PAR2-KO mice exhibited a significantly reduced tumor size when compared to their WT counterparts. Ratio of spleen weight to body weight. (C) Macroscopic discrepancies between the animal strains were observed in the ratio of spleen weight to body weight. Compared to WT (white) animals, PAR2-KO (black) animals showed a significantly higher spleen weight in relation to body weight. Apixaban concentration. (D) Apixaban plasma concentrations measured by UPLC-MS/MS in WT (white) and PAR2-KO mice (black) treated with 50 mg/kg or w/o (control) Apixaban. (E) Significantly higher Apixaban levels could also be detected in the tumors of treated animals. PCR analyzes. (F) Apixaban is metabolized via CYP3A4. The murine counterpart is CYP3A11. PCR analyses show that the treatment of the animals with Apixaban significantly increases the expression of CYP3A11 in the liver at RNA level in both, WT (white) and PAR2-KO mice (black). Further Specific RNA analyses of the removed spleens demonstrate elevated expression levels of the following genes in PAR2-KO mice: MMP2 (G) , MMP9 (H) , COX2 (I) , PAR1 (J) , EGFR (L) . PAR2 can only be detected in WT mice (K) . N = 3-6 (I-VI), Shown is the relative expression of the target normalized to Gapdh and relative to the control approaches. Mean ± SD of n independent experiments is presented for all data. One-way ANOVA, Dunnett posthoc test, p < 0.05 (*).

Article Snippet: After blocking [Li-cor, Cat. #927-60001], membranes were incubated overnight at 4°C under rotating conditions with the following primary antibodies: AKT (1:1000) [Cell Signaling, Cat. #4691], phospho-AKT (1:1000) [Cell Signaling, Cat. #4060], p38 (1:500) [Cell Signaling, Cat. #8690], phospho-p38 (1:500) [Cell Signaling, Cat. #4511], p44/42 (1:1000) [Cell Signaling, Cat. #4695], phospho-p44/42 (1:1000) [Cell Signaling, Cat. #4370], PAR2 (1:200) [Santa Cruz Biotechnologies, Cat. #sc-13504], PAR1 (1:200) [Santa Cruz Biotechnologies, Cat. #13503].

Techniques: Control, Concentration Assay, Clinical Proteomics, Tandem Mass Spectroscopy, Expressing

Journal: Journal of ovarian research

Article Title: Mast cell tryptase-PAR2 axis promotes ovarian fibrosis through RNF152-mediated stabilization of Bcl-xL.

doi: 10.1186/s13048-025-01704-4

Figure Lengend Snippet: Fig. 4 Inhibition of PAR2 alleviated tryptase-induced COL1A1 and COL1A2. (A) Immunofluorescence staining of PAR2 (red) and nuclei (blue) in ovarian theca-stroma cells. Scale bar = 50 μm. (B) Effect of tryptase (10nM, 24 h) on the protein levels of COL1A1 and COL1A2 in the presence or absence of siRNA-mediated knockdown of PAR2. (C-D) Quantitative analysis of COL1A1 and COL1A2 protein levels (n = 3). *P < 0.05 vs. Control. Data are presented as mean ± SEM

Article Snippet: After washing with phosphate-buffered saline (PBS), stromal cells were blocked with bovine serum for 1 h and incubated with primary antibodies against PAR2 (1:50, Santa Cruz, USA) and vimentin (1:100, Santa Cruz, USA) overnight at 4 °C.

Techniques: Inhibition, Immunofluorescence, Staining, Knockdown, Control

Journal: Journal of ovarian research

Article Title: Mast cell tryptase-PAR2 axis promotes ovarian fibrosis through RNF152-mediated stabilization of Bcl-xL.

doi: 10.1186/s13048-025-01704-4

Figure Lengend Snippet: Fig. 5 Tryptase/PAR2 regulated COL1A1 and COL1A2 by Bcl-xL. (A-B) Primary mouse ovarian theca-stroma cells were treated with vehicle control (0) and tryptase (1 and 10nM) for 24 h. Representative Western blot (A) and quantitative analysis (B) of Bcl-xL protein level (n = 3). (C-E) Effect of tryptase (10nM, 24 h) on the protein levels of COL1A1 and COL1A2 in the presence or absence of siRNA-mediated knockdown of Bcl-xL (n = 3). (F-J) Primary mouse ovarian theca-stroma cells were treated with 100µM PAR2 activating peptide (PAR2-AP) for 2 h or transfected with si-PAR2 for 24 h. (F) The mRNA level of Bcl-xL was measured by RT-PCR (n = 3). Representative Western blot (G, I) and quantitative analysis (H, J) of Bcl-xL protein level (n = 3). *P < 0.05 vs. Control. #P < 0.05 vs. tryptase + si-NC. Data are presented as mean ± SEM

Article Snippet: After washing with phosphate-buffered saline (PBS), stromal cells were blocked with bovine serum for 1 h and incubated with primary antibodies against PAR2 (1:50, Santa Cruz, USA) and vimentin (1:100, Santa Cruz, USA) overnight at 4 °C.

Techniques: Control, Western Blot, Knockdown, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of ovarian research

Article Title: Mast cell tryptase-PAR2 axis promotes ovarian fibrosis through RNF152-mediated stabilization of Bcl-xL.

doi: 10.1186/s13048-025-01704-4

Figure Lengend Snippet: Fig. 6 Knockdown of RNF152 reversed Tryptase-induced collagen expression. (A-B) Ovarian theca-stroma cells were transfected with scramble siRNA (si-NC), si-PAR2, or si-RNF152. Representative Western blot (A) and quantitative analysis (B) of Bcl-xL protein level (n = 3). *P < 0.01 vs. Control. ##<0.01 vs. si-RNF152. (C-E) Effect of tryptase (10nM, 24 h) on the protein levels of COL1A1 and COL1A2 in the presence or absence of siRNA-mediated knockdown of Bcl-xL or/and si-RNF152. (C) Representative Western blot of for the expression of proteins COL1A1 and COL1A2. (D-E) Quantitative analysis of COL1A1 and COL1A2 protein levels (n = 3). *P < 0.01 vs. Control. ##<0.01 vs. tryptase + si-Bcl-xL. Data are presented as mean ± SEM

Article Snippet: After washing with phosphate-buffered saline (PBS), stromal cells were blocked with bovine serum for 1 h and incubated with primary antibodies against PAR2 (1:50, Santa Cruz, USA) and vimentin (1:100, Santa Cruz, USA) overnight at 4 °C.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Control