Journal: The Journal of investigative dermatology

Article Title: PAR2 mediates itch via TRPV3 signaling in keratinocytes

doi: 10.1016/j.jid.2020.01.012

Figure Lengend Snippet: (a) Acute itch behaviors of Trpv3−/− mice and their WT littermates induced by intradermal injections of SLIGRL (50 μg), trypsin (100 μg), 2fly (10 μg), histamine (200 μg), and BAM 8-22 (150 μg). Unpaired t tests. n = 6–8. (b) WT mice were pre-injected with vehicle (Veh) (8% DMSO), 74a (150 μg, i.d.) or FSLLRY (100 μg, i.d.) 15 min before testing the scratching behaviors elicited by SLIGRL, trypsin, or 2fly. One-way ANOVA followed by Tukey’s post hoc. n = 6-8 mice per group. (c) TRPV3 expression in the skin and DRG of Trpv3+/+ and Trpv3−/− mice as determined by western blot. (d) Double immunofluorescence staining of PAR2 (green) and TRPV3 (red) in mouse keratinocytes. Scale bar, 20 μm. (e) Venn diagram showing the overlapping of PAR2 and TRPV3 in mouse keratinocytes. *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean ± s.e.m.

Article Snippet: Immunofluorescence Primary antibodies used for immunofluorescence of cultured primary keratinocytes were: anti-TRPV3 antibody (Alomone Labs, 1:300) and anti-PAR2 antibody (Santa Cruz, 1:100).

Techniques: Injection, Expressing, Western Blot, Double Immunofluorescence Staining

Journal: The Journal of investigative dermatology

Article Title: PAR2 mediates itch via TRPV3 signaling in keratinocytes

doi: 10.1016/j.jid.2020.01.012

Figure Lengend Snippet: (a) The percentages of responding keratinocytes from WT and Trpv3−/− mice stimulated with histamine (100 μM) and SLIGRL (50 μM). ***p < 0.001, unpaired t tests. n = 3 mice per group. (b) Representative fluorescence images of Fura2 (2 μM)-loaded WT, Trpv3−/− and Par2−/− keratinocytes stimulated with SLIGRL (50 μM). Scale bar, 20 μm. (c) Representative traces showing intracellular Ca2+ responses elicited by SLIGRL (50 μM) in WT mouse keratinocytes. (d, e) The effect of FSLLRY (100 μM) (d) and 74a (100 μM) (e) on SLIGRL-induced Ca2+ responses in WT mouse keratinocytes. (f) The effect of 74a (100 μM) on SLIGKV-induced (100 μM) Ca2+responses in human keratinocytes. (g) Quantified data showing the percentages of SLIGRL-responsive keratinocytes before and after the incubation of FSLLRY or 74a. ***p < 0.001, unpaired t test, n = 3 mice per group. Data are presented as mean ± s.e.m.

Article Snippet: Immunofluorescence Primary antibodies used for immunofluorescence of cultured primary keratinocytes were: anti-TRPV3 antibody (Alomone Labs, 1:300) and anti-PAR2 antibody (Santa Cruz, 1:100).

Techniques: Fluorescence, Incubation

Journal: The Journal of investigative dermatology

Article Title: PAR2 mediates itch via TRPV3 signaling in keratinocytes

doi: 10.1016/j.jid.2020.01.012

Figure Lengend Snippet: (a-c) Representative traces showing that Ca2+ transients elicited by SLIGRL (50 μM) in WT mouse keratinocytes were completely inhibited by co-incubation with 100 μM gallein, a Gβγ-protein inhibitor (a) and 10 μM U73122, a PLC inhibitor (b), but were partially inhibited by co-incubation with 10 μM DBHQ, an ATPase inhibitor (c). (d) Quantified data showing the percentages of SLIGRL-responsive keratinocytes before and after the incubation of gallein, U73122 or DBHQ. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test, n = 3 mice per group. (e) TSLP in cultured media after SLIGRL stimulation was assayed by ELISA. Keratinocytes from Trpv3−/− and Par2−/− mice released less TSLP in response to SLIGRL stimulation. n = 3 mice per group. *p < 0.05, **p < 0.01, two-way ANOVA with Bonferroni post hoc test.

Article Snippet: Immunofluorescence Primary antibodies used for immunofluorescence of cultured primary keratinocytes were: anti-TRPV3 antibody (Alomone Labs, 1:300) and anti-PAR2 antibody (Santa Cruz, 1:100).

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of investigative dermatology

Article Title: PAR2 mediates itch via TRPV3 signaling in keratinocytes

doi: 10.1016/j.jid.2020.01.012

Figure Lengend Snippet: (a) WT, Trpv3−/− and Par2−/− mice were treated daily with a topical application of MC-903. Scratching numbers were recorded on day 1, 3, 5, and 7. Two-way ANOVA followed by Bonferroni post hoc. n = 6–8 mice per group. (b) Real time RT-PCR showing the relative levels of TRPV3 and PAR2 mRNA in pruritic skin lesions of AD patients and normal control. Unpaired t test. n = 12. (c, d) Relative levels of PAR2 mRNA (c) and TRPV3 mRNA (e) in the ears of MC-903-treated mice on day 5. Two-way ANOVA with Bonferroni post hoc. n = 4–5. Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Immunofluorescence Primary antibodies used for immunofluorescence of cultured primary keratinocytes were: anti-TRPV3 antibody (Alomone Labs, 1:300) and anti-PAR2 antibody (Santa Cruz, 1:100).

Techniques: Quantitative RT-PCR

Journal: The Journal of investigative dermatology

Article Title: PAR2 mediates itch via TRPV3 signaling in keratinocytes

doi: 10.1016/j.jid.2020.01.012

Figure Lengend Snippet: (a-d) Ear appearance of WT mice topically applied with vehicle as a control (a), MC-903 (b), MC-903+74a (c), and MC-903+FSLLRY (d) for 7 days. (e-h) Hematoxylin-eosin staining showing changes in hyperkeratosis and inflammatory infiltration in the ears of corresponding mice in panels a-d, respectively. Scale = 50 μm. (i. j) The scratching numbers (i) and ear thickness increment (j) of mice treated with MC-903, MC-903+74a and MC-903+FSLLRY for 7 days. One-way ANOVA followed by Tukey’s post hoc. n = 6–8 mice per group. (k) Diagram illustrating PAR2-TRPV3 signaling pathway in itch.

Article Snippet: Immunofluorescence Primary antibodies used for immunofluorescence of cultured primary keratinocytes were: anti-TRPV3 antibody (Alomone Labs, 1:300) and anti-PAR2 antibody (Santa Cruz, 1:100).

Techniques: Staining

Journal: The Journal of investigative dermatology

Article Title: PAR2 mediates itch via TRPV3 signaling in keratinocytes

doi: 10.1016/j.jid.2020.01.012

Figure Lengend Snippet: (a) Acute itch behaviors of Trpv3−/− mice and their WT littermates induced by intradermal injections of SLIGRL (50 μg), trypsin (100 μg), 2fly (10 μg), histamine (200 μg), and BAM 8-22 (150 μg). Unpaired t tests. n = 6–8. (b) WT mice were pre-injected with vehicle (Veh) (8% DMSO), 74a (150 μg, i.d.) or FSLLRY (100 μg, i.d.) 15 min before testing the scratching behaviors elicited by SLIGRL, trypsin, or 2fly. One-way ANOVA followed by Tukey’s post hoc. n = 6-8 mice per group. (c) TRPV3 expression in the skin and DRG of Trpv3+/+ and Trpv3−/− mice as determined by western blot. (d) Double immunofluorescence staining of PAR2 (green) and TRPV3 (red) in mouse keratinocytes. Scale bar, 20 μm. (e) Venn diagram showing the overlapping of PAR2 and TRPV3 in mouse keratinocytes. *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean ± s.e.m.

Article Snippet: The primary antibodies used were goat anti-TRPV3 (Alomone Labs, 1:100) and goat anti-GAPDH (Leagene, 1:1000) antibodies.

Techniques: Injection, Expressing, Western Blot, Double Immunofluorescence Staining

Journal: The Journal of investigative dermatology

Article Title: PAR2 mediates itch via TRPV3 signaling in keratinocytes

doi: 10.1016/j.jid.2020.01.012

Figure Lengend Snippet: (a) The percentages of responding keratinocytes from WT and Trpv3−/− mice stimulated with histamine (100 μM) and SLIGRL (50 μM). ***p < 0.001, unpaired t tests. n = 3 mice per group. (b) Representative fluorescence images of Fura2 (2 μM)-loaded WT, Trpv3−/− and Par2−/− keratinocytes stimulated with SLIGRL (50 μM). Scale bar, 20 μm. (c) Representative traces showing intracellular Ca2+ responses elicited by SLIGRL (50 μM) in WT mouse keratinocytes. (d, e) The effect of FSLLRY (100 μM) (d) and 74a (100 μM) (e) on SLIGRL-induced Ca2+ responses in WT mouse keratinocytes. (f) The effect of 74a (100 μM) on SLIGKV-induced (100 μM) Ca2+responses in human keratinocytes. (g) Quantified data showing the percentages of SLIGRL-responsive keratinocytes before and after the incubation of FSLLRY or 74a. ***p < 0.001, unpaired t test, n = 3 mice per group. Data are presented as mean ± s.e.m.

Article Snippet: The primary antibodies used were goat anti-TRPV3 (Alomone Labs, 1:100) and goat anti-GAPDH (Leagene, 1:1000) antibodies.

Techniques: Fluorescence, Incubation

Journal: The Journal of investigative dermatology

Article Title: PAR2 mediates itch via TRPV3 signaling in keratinocytes

doi: 10.1016/j.jid.2020.01.012

Figure Lengend Snippet: (a-c) Representative traces showing that Ca2+ transients elicited by SLIGRL (50 μM) in WT mouse keratinocytes were completely inhibited by co-incubation with 100 μM gallein, a Gβγ-protein inhibitor (a) and 10 μM U73122, a PLC inhibitor (b), but were partially inhibited by co-incubation with 10 μM DBHQ, an ATPase inhibitor (c). (d) Quantified data showing the percentages of SLIGRL-responsive keratinocytes before and after the incubation of gallein, U73122 or DBHQ. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test, n = 3 mice per group. (e) TSLP in cultured media after SLIGRL stimulation was assayed by ELISA. Keratinocytes from Trpv3−/− and Par2−/− mice released less TSLP in response to SLIGRL stimulation. n = 3 mice per group. *p < 0.05, **p < 0.01, two-way ANOVA with Bonferroni post hoc test.

Article Snippet: The primary antibodies used were goat anti-TRPV3 (Alomone Labs, 1:100) and goat anti-GAPDH (Leagene, 1:1000) antibodies.

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of investigative dermatology

Article Title: PAR2 mediates itch via TRPV3 signaling in keratinocytes

doi: 10.1016/j.jid.2020.01.012

Figure Lengend Snippet: (a) WT, Trpv3−/− and Par2−/− mice were treated daily with a topical application of MC-903. Scratching numbers were recorded on day 1, 3, 5, and 7. Two-way ANOVA followed by Bonferroni post hoc. n = 6–8 mice per group. (b) Real time RT-PCR showing the relative levels of TRPV3 and PAR2 mRNA in pruritic skin lesions of AD patients and normal control. Unpaired t test. n = 12. (c, d) Relative levels of PAR2 mRNA (c) and TRPV3 mRNA (e) in the ears of MC-903-treated mice on day 5. Two-way ANOVA with Bonferroni post hoc. n = 4–5. Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The primary antibodies used were goat anti-TRPV3 (Alomone Labs, 1:100) and goat anti-GAPDH (Leagene, 1:1000) antibodies.

Techniques: Quantitative RT-PCR

Journal: The Journal of investigative dermatology

Article Title: PAR2 mediates itch via TRPV3 signaling in keratinocytes

doi: 10.1016/j.jid.2020.01.012

Figure Lengend Snippet: (a-d) Ear appearance of WT mice topically applied with vehicle as a control (a), MC-903 (b), MC-903+74a (c), and MC-903+FSLLRY (d) for 7 days. (e-h) Hematoxylin-eosin staining showing changes in hyperkeratosis and inflammatory infiltration in the ears of corresponding mice in panels a-d, respectively. Scale = 50 μm. (i. j) The scratching numbers (i) and ear thickness increment (j) of mice treated with MC-903, MC-903+74a and MC-903+FSLLRY for 7 days. One-way ANOVA followed by Tukey’s post hoc. n = 6–8 mice per group. (k) Diagram illustrating PAR2-TRPV3 signaling pathway in itch.

Article Snippet: The primary antibodies used were goat anti-TRPV3 (Alomone Labs, 1:100) and goat anti-GAPDH (Leagene, 1:1000) antibodies.

Techniques: Staining