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Amadeus S. Zhu, Ang Li, Tabetha S. Ratliff, Martha Melsom, Luis A

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Presentation on theme: "Amadeus S. Zhu, Ang Li, Tabetha S. Ratliff, Martha Melsom, Luis A"— Presentation transcript:

1 After Skin Wounding, Noncoding dsRNA Coordinates Prostaglandins and Wnts to Promote Regeneration 
Amadeus S. Zhu, Ang Li, Tabetha S. Ratliff, Martha Melsom, Luis A. Garza  Journal of Investigative Dermatology  Volume 137, Issue 7, Pages (July 2017) DOI: /j.jid Copyright © 2017 The Authors Terms and Conditions

2 Figure 1 Poly(I:C) induces WNT7B expression in wounds and cultured NHEKs. (a) Representative confocal scanning light microscopy image of regenerated hair follicles in C57BL6/NJ mouse wounds injected with or without poly(I:C). (b) Wnt ligands most strongly up-regulated or down-regulated in gene expression microarray comparing NHEKs treated with vehicle or poly(I:C), published under GSE (c) Representative immunofluorescent image of Wnt7b protein in C57BL6/NJ mouse wounds. Scale bar = 100 μm. (d) Quantitative real-time reverse transcriptase–PCR analysis of WNT7B mRNA expression in NHEKs (n = 3) treated with various doses of poly(I:C). (e) Western blot analysis of Wnt7b protein expression in NHEKs treated with or without poly(I:C), with β-actin as a loading control. NHEK, normal human epidermal keratinocyte; poly(I:C), polyinosinic:polycytidylic acid; SE, standard error. Journal of Investigative Dermatology  , DOI: ( /j.jid ) Copyright © 2017 The Authors Terms and Conditions

3 Figure 2 WNT7B expression is regulated through the TLR3/IL-6/STAT3 axis. (a) Quantitative real-time reverse transcriptase–PCR analysis of Wnt7b mRNA expression in background (C57BL6/NJ) and TLR3-KO mouse wounds (n = 8). (b) Wnt7b mRNA expression in TLR3-KO mouse wounds injected with vehicle or recombinant IL-6 (n = 8). (c) Wnt7b mRNA expression in background or STAT3-KO mouse wounds (n = 8). (d) WNT7B mRNA expression in NHEKs transfected with mock or TLR3-specific siRNA, followed by treatment with or without poly(I:C). ∗P < KO, knockout; NHEK, normal human epidermal keratinocyte; poly(I:C), polyinosinic:polycytidylic acid; SE, standard error; siRNA, small interfering RNA; STAT, signal transducer and activator of transcription; TLR, toll-like receptor; WT, wild type. Journal of Investigative Dermatology  , DOI: ( /j.jid ) Copyright © 2017 The Authors Terms and Conditions

4 Figure 3 Poly(I:C) induces PTGS2 expression in wounds and cultured NHEKs. (a) Quantitative real-time reverse transcriptase–PCR analysis of PTGS2 mRNA expression in mouse wounds at PWDs 1, 3, and 5, normalized to pre-wound levels. (b) PTGS2 mRNA expression in NHEKs treated with various doses of poly(I:C). ∗P < (c) PTGDS and PTGES mRNA expression in NHEKs treated with poly(I:C). (d) PTGS1/2 and HPGD in gene expression microarray in Figure 1b. HPGD, 15-hydroxyprostaglandin dehydrogenase; NHEK, normal human epidermal keratinocyte; poly(I:C), polyinosinic:polycytidylic acid; PTGDS, prostaglandin D2 synthase; PTGES, prostaglandin E2 synthase; PTGS, prostaglandin synthase; PWD, post-wounding day. Journal of Investigative Dermatology  , DOI: ( /j.jid ) Copyright © 2017 The Authors Terms and Conditions

5 Figure 4 PTGS2 is required for poly(I:C)-induced WNT7B expression. (a) PTGS2-knockdown efficiency in NHEKs transfected with mock or PTGS2-specific siRNA. (b) Quantitative real-time reverse transcriptase–PCR analysis of WNT7B mRNA expression in NHEKs (n = 3) transfected with mock or PTGS2-specific siRNA, followed by treatment with or without poly(I:C), with the addition of PGE2, PGF2α, or 11b-PGF2α. ‡PGD2 induced significant cytotoxicity. (c) Representative Western blot of WNT7B protein expression in NHEKs treated as in b (n = 3). (d) Western blot quantification for n = 3 repeats performed as described in c. (e) Representative confocal scanning light microscopy image of regenerated hair follicles in mouse wounds injected with poly(I:C), with or without celecoxib. (f) Quantification of hair follicles in confocal scanning light microscopy images from d (n = 15–17). (g) Representative confocal scanning light microscopy image of regenerated hair follicles in mouse wounds injected with poly(I:C) and celecoxib, with or without dmPGE2. (h) Quantification of hair follicles in confocal scanning light microscopy images from e (n = 10–11). ∗P < CEL, celecoxib; dmPGE2, 16,16-dimethyl PGE2; NHEK, normal human epidermal keratinocyte; poly(I:C), polyinosinic:polycytidylic acid; PG, prostaglandin; PIC, polyinosinic:polycytidylic acid; PTGS, prostaglandin synthase; siRNA, small interfering RNA. Journal of Investigative Dermatology  , DOI: ( /j.jid ) Copyright © 2017 The Authors Terms and Conditions

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