IgE induces transcriptional regulation of thymic stromal lymphopoietin in human airway smooth muscle cells Naresh Singh Redhu, MSc, Ali Saleh, PhD, Hai-Chon.

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IgE induces transcriptional regulation of thymic stromal lymphopoietin in human airway smooth muscle cells Naresh Singh Redhu, MSc, Ali Saleh, PhD, Hai-Chon Lee, PhD, Andrew J. Halayko, PhD, Steven F. Ziegler, PhD, Abdelilah S. Gounni, PhD Journal of Allergy and Clinical Immunology Volume 128, Issue 4, Pages 892-896.e2 (October 2011) DOI: 10.1016/j.jaci.2011.06.045 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 TSLP mRNA (A) and protein expression (B) were measured from ASM cells stimulated with IgE or TNF-α by using real-time RT-PCR and ELISA, respectively. C, ASM cells were transiently transfected with human TSLP promoter luciferase construct and stimulated with IgE or TNF-α in the presence of a Syk inhibitor, R112, or vehicle (dimethyl sulfoxide). TSLP promoter activity was measured. n = 4, ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 compared with the unstimulated (Fig 1, A, Mann-Whitney test; Fig 1, B, unpaired t test) or stimulated (Fig 1, C, unpaired t test) control. Journal of Allergy and Clinical Immunology 2011 128, 892-896.e2DOI: (10.1016/j.jaci.2011.06.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 A, Lentiviral transduction efficiency in ASM for the Syk-shRNAs as determined by FACS by using turbo-green fluorescent protein as a marker. B, Lentiviral Syk-shRNA transduction silenced the ASM Syk expression efficiently as observed by Western blotting. C, Syk-silenced and scramble ASM cells were transfected with a TSLP promoter construct, stimulated with IgE or TNF-α for 12 hours, and TSLP promoter activity was measured. n = 3, ∗P < .05, ∗∗P < .01 (Fig 2, C, unpaired t test) compared with the unstimulated control. Journal of Allergy and Clinical Immunology 2011 128, 892-896.e2DOI: (10.1016/j.jaci.2011.06.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Primary ASM cells were transfected with (A) mutant constructs of NF-κB (M1), AP-1 (M2), or WT TSLP promoter (WT); (B) WT TSLP promoter construct and NF-κB/p65 subunit overexpressing plasmid; and (C) WT TSLP promoter and dnIKKβ. Cells were stimulated with IgE or TNF-α for 12 hours, and luciferase reporter activity was measured. n = 3, ∗P < .05, ∗∗P < .01 (Fig 3, A, Mann-Whitney test; Fig 3, B and C, unpaired t test) compared with the stimulated WT control. Journal of Allergy and Clinical Immunology 2011 128, 892-896.e2DOI: (10.1016/j.jaci.2011.06.045) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions