Journal: Pain Reports

Article Title: Monoclonal antibody inhibition of PAR2 reduces phenotype severity and pain in murine inflammatory bowel disease

doi: 10.1097/PR9.0000000000001446

Figure Lengend Snippet: Colonic PAR2 activation by 2F elicits LSN responses that are mediated by endosomal internalization, PKA and PKC. (A) Experimental framework for LSN recordings showing a cannulated colon in a recording chamber with the LSN aspirated into a suction electrode. (B) Representative action potentials recorded from LSN afferent fiber prestimulation. (C and D) 100 µM 2F application elicits LSN responses illustrated by (C) action potential traces prestimulation and 15 minutes poststimulation and (D) change in LSN firing rate over time after stimulation via the luminal inflow (vertical dotted line). 2F-stimulation increased LSN firing (E and F). Timeline and peak change in firing rate after 100 µM 2F application (vertical dotted line) in tissue pretreated with (E) 50 µM PitStop2 (PS2) inhibitor for endosomal internalization or negative control PitNot2 (PN2) and (F) 100 µM H-89 dihydrochloride (H-89) and bisindolylmaleimide (GFX), PKA and PKC inhibitors or DMSO vehicle. (E) PS2 as well as separate and simultaneous pretreatment with H-89 and GFX reduced the peak response to 2F. (D and E) Independent samples t test comparing the peak change in firing rate (N = 5–9). (F) One-way ANOVA with post-hoc FDR-corrected independent samples t test (N = 5–6). * P < 0.05, ** P < 0.01, *** P < 0.001 **** P < 0.0001. Data are presented as mean ± SD. LSN, lumbar splanchnic nerve; PAR2, protease-activated receptor 2; PKA, protein kinase A; PKC, protein kinase C.

Article Snippet: When measuring the inhibitory concentration 50 (IC50), cells received PAR650097 mIgG or hIgG or isotype control antibodies, for 60 minutes at room temperature before addition of PAR2 activating matriptase (3946-SEB-010, R&D Systems, Abingdon, United Kingdom).

Techniques: Activation Assay, Negative Control

Journal: Pain Reports

Article Title: Monoclonal antibody inhibition of PAR2 reduces phenotype severity and pain in murine inflammatory bowel disease

doi: 10.1097/PR9.0000000000001446

Figure Lengend Snippet: PAR2 activation in the colon by 2F sensitizes LSN responses to mechanical and chemical stimulation of the colon via endosomal internalization, PKA and PKC. LSN action potential firing after mechanical distention: gradually increasing intraluminal pressure from 0 to 80 mm Hg, and chemical application: 1 mM cinnamaldehyde and 1 µM capsaicin are illustrated in (A). (B) Responses to distention were quantified through the change in firing rate over increasing discrete pressure values. (C and D) Responses to chemical stimuli were quantified through the change in firing rate over time after application (vertical dotted line). 100 µM 2F stimulation, but not vehicle control, elicited sensitization of the LSN to (B) distention, (C) cinnamaldehyde, and (D) capsaicin. Pretreatment with PS2 before 2F stimulation reduced subsequent LSN responses to distention, cinnamaldehyde, and capsaicin compared with PN2. Pretreatment with H-89 and GFX, applied either simultaneously or individually, also reduced subsequent peak LSN responses to distention, cinnamaldehyde, and capsaicin compared with DMSO vehicle. (B–D) Independent samples t tests and one-way ANOVA with post-hoc FDR-corrected independent samples t test (N = 5–9). * P < 0.05, ** P < 0.01, **** P < 0.0001. Data are presented as means ± SD. LSN, lumbar splanchnic nerve; PAR2, protease-activated receptor 2; PKA, protein kinase A; PKC, protein kinase C.

Article Snippet: When measuring the inhibitory concentration 50 (IC50), cells received PAR650097 mIgG or hIgG or isotype control antibodies, for 60 minutes at room temperature before addition of PAR2 activating matriptase (3946-SEB-010, R&D Systems, Abingdon, United Kingdom).

Techniques: Activation Assay, Control

Journal: iScience

Article Title: PAR2-β-arrestin 2-ERK axis mediates Malassezia globosa -induced IL-17 response by disrupting ZO-1 in keratinocytes

doi: 10.1016/j.isci.2026.115646

Figure Lengend Snippet: Expression of PAR2, IL-17, and TJ proteins in Malassezia folliculitis (A and B) PAR2, IL-17, ZO-1, occludin, and claudin-1 were validated in MF and CTRL skin tissue by immunohistochemical staining (IHC). Values are mean ± SD of six samples from two independent experiments. Statistical analysis was performed using a two-way ANOVA with Šídák’s multiple comparisons test (∗ p < 0.05; ∗∗∗ p < 0.001). (C and D) The spores rate of CTRL and MF groups by Periodic Acid-Schif staining. Purple arrows indicate the spores. Scale bars, 50 μm. Data are presented as mean ± SD of triplicate wells from three independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t tests (∗∗ p < 0.01).

Article Snippet: Primer of PAR2 (Forward, 5′-GGCAC TCCAGGAAGAAGGCAAAC-3’; Reverse, 5′-CAGGGCAGGAATGAAGATGGTCTG-3′) , Sangon Biotech , N/A.

Techniques: Expressing, Immunohistochemical staining, Staining, Two Tailed Test

Journal: iScience

Article Title: PAR2-β-arrestin 2-ERK axis mediates Malassezia globosa -induced IL-17 response by disrupting ZO-1 in keratinocytes

doi: 10.1016/j.isci.2026.115646

Figure Lengend Snippet: Impact of M. globosa infection on PAR2 activation, IL-17 production, and TJ integrity in HaCaT cells (A) Cell survival rate was detected by CCK8 assay. (B–D) Protein expression and correlation analysis in HaCaT cells infected with 30 MOI M. globosa was evaluated by western blot at various time. (E and F) mRNA levels and correlation analysis were measured by reverse transcription quantitative PCR (RT-qPCR). Red indicated upregulated gene expression, green indicated downregulated gene expression. (G) IL-17A levels were detected by ELISA test in the supernatant of HaCaT cells infected with 30 MOI M. globosa . Red indicated upregulated gene expression (C and E) or positive correlation (D and F), blue indicated downregulated gene expression (C and E) or negative correlation (D and F).These experiments (A, B, E, and G) were representative of 3 independent experiments. Values are means ± SD. a two-way ANOVA with Bonferroni’s (A), or Dunnet’s (G) multiple comparisons test was applied. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Correlation analysis is performed using Pearson’s correlation coefficient analysis. See also .

Article Snippet: Primer of PAR2 (Forward, 5′-GGCAC TCCAGGAAGAAGGCAAAC-3’; Reverse, 5′-CAGGGCAGGAATGAAGATGGTCTG-3′) , Sangon Biotech , N/A.

Techniques: Infection, Activation Assay, CCK-8 Assay, Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: PAR2-β-arrestin 2-ERK axis mediates Malassezia globosa -induced IL-17 response by disrupting ZO-1 in keratinocytes

doi: 10.1016/j.isci.2026.115646

Figure Lengend Snippet: Activation of PAR2-β-arrestin 2-ERK signaling axis and IL-17 in M. globosa -infected HaCaT cells (A and B) Protein expression level following PAR2 antagonist intervention using western blotting. (C and D) PAR2-related pathway proteins expression following siRNA transfection by western blotting. (E) IL-17 levels in cell supernatants after siRNA transfection by ELISA. (F and G) Fluorescence of JC-1 aggregates to monomers. Scale bars, 10 μm. These experiments (A, C, E, and F) were representative of 3 independent experiments. Values are mean ± SD. Statistical analysis was performed using a two-way ANOVA with Šídák’s multiple comparisons test (ns, no statistical significance; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). See also .

Article Snippet: Primer of PAR2 (Forward, 5′-GGCAC TCCAGGAAGAAGGCAAAC-3’; Reverse, 5′-CAGGGCAGGAATGAAGATGGTCTG-3′) , Sangon Biotech , N/A.

Techniques: Activation Assay, Infection, Expressing, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: iScience

Article Title: PAR2-β-arrestin 2-ERK axis mediates Malassezia globosa -induced IL-17 response by disrupting ZO-1 in keratinocytes

doi: 10.1016/j.isci.2026.115646

Figure Lengend Snippet: PAR2 interacts with β-arrestin 2 and ZO-1 in M. globosa -infected HaCaT cells (A and B) Expression and co-localization of PAR2, β-arrestin 2, and ZO-1 in HaCaT cells by immunofluorescence. White arrows: co-localization. Scale bars, 10 μm. (C and D) Expression and co-localization of PAR2 and ZO-1 by PLA. The yellow arrows indicated the PLA-positive signal, Scale bars, 20 μm. (E and F) Interaction regulation of PAR2, β-arrestin 2, and ZO-1 using CoIP. These experiments (A, C, and E) were representative of three independent experiments. Data are presented as mean ± SD. Statistical analysis was performed using a two-way ANOVA with Šídák’s multiple comparisons test (ns, no statistical significance; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). (G) PAR2 interaction with ZO-1 and β-arrestin 2 using MbYTH. G1. MbYTH of PAR2 and control NR1I2. (a) Positive control, (b) negative control, (c) experimental groups: pDHB1-PAR2 and pPR3-N-NR1I2, (d) autoactivation 1: pDHB1-PAR2, and (e) autoactivation 2: pPR3-N-NR1I2. G2. MbYTH of PAR2 and ZO-1. (a) Positive control, (b) negative control, (c) experimental groups: pDHB1-PAR2 and pPR3-N-ZO-1, (d) autoactivation 1: pDHB1-PAR2, and (e) autoactivation 2: pPR3-N-ZO-1. G3. MbYTH of PAR2 and β-arrestin 2. (a) Positive control, (b) negative control, (c) experimental groups: pDHB1-PAR2 and pPR3-N-β-arrestin 2, (d) autoactivation: pPR3-N-β-arrestin 2. SD/-Leu/-Trp medium; SD/-Leu/-Trp/-His medium containing 5 mM 3-AT; SD/-Leu/-Trp/-His/-Ade medium; SD/-Leu/-Trp/-His/-Ade medium containing 5 mM 3-AT. 1/2/3/4 were 10,000/1,000/100/10 times dilution of the original bacterial solution.

Article Snippet: Primer of PAR2 (Forward, 5′-GGCAC TCCAGGAAGAAGGCAAAC-3’; Reverse, 5′-CAGGGCAGGAATGAAGATGGTCTG-3′) , Sangon Biotech , N/A.

Techniques: Infection, Expressing, Immunofluorescence, Control, Positive Control, Negative Control

Journal: iScience

Article Title: PAR2-β-arrestin 2-ERK axis mediates Malassezia globosa -induced IL-17 response by disrupting ZO-1 in keratinocytes

doi: 10.1016/j.isci.2026.115646

Figure Lengend Snippet: Attenuation of PAR2-β-arrestin 2-ERK signaling and IL-17 responses in Par2 -/- mice with skin M. globosa infection (A) Skin lesions and pathological staining of mouse skin. Scale bars, 50 μm. (B and C) Expression level of PAR2, IL-17, ZO-1 by IHC. (D and E) Expression and co-localization of PAR2, β-arrestin 2, and ZO-1 by immunofluorescence. White dotted line indicated the basement membrane zone. Scale bars, 50 μm. (F and G) Expression of related proteins using western blotting. (H) IL-17 levels using ELISA. (B, C, and H) For each group, n = 6. These experiments (A, D, E, F, and G) were representative of 3 independent experiments. Data are presented as mean ± SD. Statistical significance was determined using a two-way ANOVA with Šídák’s multiple comparisons test (ns, no statistical significance; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001).

Article Snippet: Primer of PAR2 (Forward, 5′-GGCAC TCCAGGAAGAAGGCAAAC-3’; Reverse, 5′-CAGGGCAGGAATGAAGATGGTCTG-3′) , Sangon Biotech , N/A.

Techniques: Infection, Staining, Expressing, Immunofluorescence, Membrane, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: PAR2-β-arrestin 2-ERK axis mediates Malassezia globosa -induced IL-17 response by disrupting ZO-1 in keratinocytes

doi: 10.1016/j.isci.2026.115646

Figure Lengend Snippet: The possible mechanism of PAR2-β-arrestin 2-ERK signaling pathway on IL-17 release in keratinocytes On physiological state, PAR2, β-arrestin 2, and ZO-1 are in resting state, and ZO-1 is crucial for skin barrier maintenance. After overgrown M. globosa invades keratinocytes, the interaction of PAR2 with β-arrestin 2 and ZO-1 is enhanced and internalized, which upregulates ERK1/2 expression and ZO-1 disruption, thus promoting intracellular expression and extracellular release of IL-17.

Article Snippet: Primer of PAR2 (Forward, 5′-GGCAC TCCAGGAAGAAGGCAAAC-3’; Reverse, 5′-CAGGGCAGGAATGAAGATGGTCTG-3′) , Sangon Biotech , N/A.

Techniques: Expressing, Disruption