1. Extracellular Double-Stranded RNA Induces TSLP via an Endosomal Acidification- and NF-κB-Dependent Pathway in Human Keratinocytes 
Anh T. Vu, Xue Chen, Yang Xie, Seiji Kamijo, Hiroko Ushio, Junko Kawasaki, Mutsuko Hara, Shigaku Ikeda, Ko Okumura, Hideoki Ogawa, Toshiro Takai 
Journal of Investigative Dermatology 
Volume 131, Issue 11, Pages (November 2011)
DOI: /jid
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2. Figure 1
Bafilomycin A1 (BFA) blocked polyinosinic-polycytidylic acid (poly(IC))-induced expression of thymic stromal lymphopoietin (TSLP) in keratinocytes. (a) Protein concentrations in the culture supernatant.*P<0.001 versus poly(IC) without the inhibitors by analysis of variance (ANOVA) with Tukey's multiple comparison test. (b) Levels of mRNA at 3 and 9 hours are presented relative to the medium at 3 hours. Data shown are the mean±SD for three wells (a) and the values for single wells (b), and are representative of three independent experiments with similar results. Keratinocytes were treated with 100 nM BFA or 5 mM 2-aminopurine (2AP) before stimulation.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Bafilomycin A1 (BFA) did not inhibit FSL-1-induced expression of thymic stromal lymphopoietin (TSLP) in keratinocytes. (a) Protein concentrations in the culture supernatant. Broken line shows the minimum detection limit.*P<0.001 versus the stimulation without the inhibitors by analysis of variance (ANOVA) with Tukey's multiple comparison test. (b) Levels of mRNA at 4, 6, and 8 hours are presented relative to the medium at 4 hours. Data shown are the mean±SD for three wells (a) and the values for single wells (b), and are representative of three independent experiments with similar results. Keratinocytes were treated with 100 nM BFA or 5 mM 2-aminopurine (2AP) before stimulation.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Transfection with RelA small interfering RNA (siRNA), but not IFN-regulatory factor 3 (IRF3) siRNA, inhibited the polyinosinic-polycytidylic acid poly(IC)-induced gene expression of thymic stromal lymphopoietin (TSLP) in keratinocytes. Keratinocytes transfected with siRNAs using LipofectamineRNAiMAX (LA-MAX) were stimulated with poly(IC). Levels of mRNA at 6 and 9 hours are presented relative to that without the stimulation at 6 and 9 hours, respectively. L and M represent 35–45% and 45–55% guanine-cytosine content, respectively. Results using RelA-siRNA2 and IRF3-siRNA3 are shown. Data shown are the values for single wells and representative of three independent experiments including those using RelA and IRF3 siRNAs with other sequences.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Bafilomycin A1 (BFA) most efficiently blocked the polyinosinic-polycytidylic acid (poly(IC))-induced release of thymic stromal lymphopoietin (TSLP) in keratinocytes. Keratinocytes were stimulated with poly(IC) purchased from GE Healthcare/Amersham ina–c. (a) 2-Aminopurine (2AP; 5 mM) and SU6668 (5 μM) did not inhibit the release of TSLP. (b) Comparison between 1 and 20 μg ml–1 poly(IC). (c) Dose dependency of the effect of 2AP and SU6668. (d) Keratinocytes were stimulated with poly(IC)s (high and low molecular weight (HMW and LMW)) purchased from InvivoGen. Protein concentrations in the culture supernatant were measured. Broken line shows the minimum detection limit.*P<0.05 versus the stimulation without the inhibitors by analysis of variance (ANOVA) with Tukey's multiple comparison test. Data shown are the mean±SD for three wells and are representative of three (a–c) or two (d) independent experiments with similar results.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Expression of the four double-stranded RNA (dsRNA) sensors in keratinocytes. Keratinocytes with (polyinosinic-polycytidylic acid (Poly(IC)) or without (Medium) stimulation with poly(IC) were analyzed. (a) mRNA expression of the dsRNA sensors at 3, 6, and 9 hours relative to the average level of TLR3 expression at 3 hours without poly(IC). Data shown are the mean±SD for three or four independent experiments. (b) Analysis of TLR3 expression by flow cytometry. Cell-surface (Surface staining) and intracellular (Intracellular staining) TLR3 were stained. Data shown are representative of three independent experiments with similar results. MDA5, melanoma differentiation antigen 5; PKR, IFN-inducible double-stranded RNA-activated protein kinase; RIG-I, retinoic acid-inducible gene-I; TLR3, Toll-like receptor 3.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Effect of bafilomycin A1 (BFA), 2-aminopurine (2AP), and SU6668 on the double-stranded RNA (dsRNA) sensor expression. (a) Effect of BFA. (b) Effect of 2AP and SU6668. Levels of mRNA at 6 hours are presented relative to the medium.*P<0.05 versus the stimulation without the inhibitors by analysis of variance (ANOVA) with Tukey's multiple comparison test. Data shown are the mean±SD for three wells and are representative of three (a) or two (b) independent experiments with similar results. Keratinocytes were treated with 100 nM BFA, 5 mM 2AP, or 5 μM SU6668 before stimulation. MDA5, melanoma differentiation antigen 5; PKR, IFN-inducible double-stranded RNA-activated protein kinase; RIG-I, retinoic acid-inducible gene-I; TLR3, Toll-like receptor 3.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions