1. Regulation of IL-33 Expression by IFN-γ and Tumor Necrosis Factor-α in Normal Human Epidermal Keratinocytes 
Jitlada Meephansan, Hidetoshi Tsuda, Mayumi Komine, Shin-ichi Tominaga, Mamitaro Ohtsuki 
Journal of Investigative Dermatology 
Volume 132, Issue 11, Pages (November 2012)
DOI: /jid
Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
IFN-γ treatment induces IL-33 mRNA and protein expression in NHEKs. (a) IL-33 mRNA expression analysis by RT-PCR. NHEKs were stimulated with IFN-γ at 0, 10, 20IUml−1 and harvested after 6hours. (b) NHEKs were harvested at the indicated time points after IFN-γ stimulation (100IUml−1); cell lysates underwent western blotting. (c) Nuclear (N) and cytosolic (C) lysates were analyzed by western blotting after 24-hour NHEK stimulation with TNF-α (100ngml−1) or IFN-γ (100IUml−1). (d) NHEKs were harvested after 24-hour stimulation with IL-4 at the indicated concentrations; cell lysates underwent western blotting. RT-PCR results are the mean±SD of three independent experiments. Representative western blotting results are shown. *P<0.05, **P<0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NHEKs, normal human epidermal keratinocytes; RT-PCR, real-time PCR; TNF-α, tumor necrosis factor-α.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Signaling molecules involved in IFN-γ-induced IL-33 expression. NHEKs were preincubated with inhibitors for (a–c) ERK (50μM PD98059), p38 (5μM SB202190), NF-κB (5μM parthenolide or 2μM BAY ), EGFR (5μM PD or PD168393), (d, e) JAK (JAK inhibitor I at indicated concentrations) for 1hour before IFN-γ stimulation (100IUml−1) for 8hours for RT-PCR (a, b, d), and 24hours for western blotting (c, e). (a, b, d) IL-33 mRNA expression examined by RT-PCR. Results are the mean±SD of three independent experiments. (c, e) Protein expression of IL-33 examined by western blotting. Loading control: α-tubulin. *P<0.05, **P<0.01. ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NHEKs, normal human epidermal keratinocytes; RT-PCR, real-time PCR.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
TNF-α plus IFN-γ induces cleavage of pro-IL-33 to mature IL-33 in NHEKs. (a) NHEKs were incubated with TNF-α (30ngml−1) plus IFN-γ at the indicated doses. Western blotting detected IL-33 (30 and 20kDa) and α-calpain 1 protein expression at 6 and 22hours. Loading control: α-tubulin. (b) NHEKs were preincubated with 50μM calpain inhibitor III, calpain inhibitor IV, caspase inhibitor I, caspase inhibitor III, or 100μM Z-VAD-FMK for 1hour before TNF-α (30ngml−1) and IFN-γ (100IUml−1) stimulation. After 24hours, cells were harvested; whole cell lysates were subjected to western blotting. Loading control: α-tubulin. The blot shown is representative of four experiments. inh, inhibitor; M, molecular weight size marker; NHEKs, normal human epidermal keratinocytes; TNF-α, tumor necrosis factor-α.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
IL-33 knockdown by siRNA reduces the expression of 20-, 25-, and 30-kDa IL-33 bands. Alpha-calpain 1 production is induced in IFN-γ and TNF-α-treated NHEKs.(a) siRNA for IL-33 and control oligos were transfected into NHEKs, cells were harvested after 48-hour incubation, total RNA was extracted, and RT-PCR evaluated IL-33 mRNA expression. A decrease in relative IL-33 mRNA expression of >90% was observed in IL-33 siRNA-transfected cells compared with control RNA-transfected cells. (b) siRNA for IL-33 and control oligos were transfected into NHEKs; after 48-hour incubation, cells were cultured in keratinocyte basal medium for 24hours. After starvation, cells were stimulated with IFN-γ (100IUml−1) and TNF-α (30ngml−1) with/without inhibitors and incubated for 24hours. Cells were harvested and analyzed by western blotting. (c) NHEKs were cultured in IFN-γ (100IUml−1) or TNF-α (30ngml−1) alone, or a combination of both cytokines. Whole cell lysates were prepared after 24-hour stimulation, and western blotting detected α-calpain 1. Loading control: α-tubulin. The blot shown is representative of three experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, molecular weight size marker; NHEKs, normal human epidermal keratinocytes; RT-PCR, real-time PCR; siRNA, small interference RNA; TNF-α, tumor necrosis factor-α.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Recombinant IL-33 induced production of IL-8 in NHEKs. IL-33 knockdown by siRNA enhanced the IL-8 induction by TNF-α. (a) Subconfluent NHEKs were stimulated with recombinant human IL-33 (50 or 100ngml−1), incubated for 24 and 48hours, and cell culture supernatants were harvested. ELISA measured the IL-8 protein. (b, c) siRNA for IL-33 or control oligos were transfected into NHEKs incubated for 24hours; cells were harvested, total RNA was extracted, and RT-PCR evaluated IL-33 mRNA (b) or IL-8 mRNA expression (c). (d) siRNA for IL-33 or control oligos were transfected, cells were incubated for 24hours, then the medium was changed to keratinocyte basal medium. After 24-hour starvation, the medium was changed into new basal medium with/without 100ngml−1 TNF-α. After 24-hour incubation, culture supernatant was collected and centrifuged to exclude cell debris. The supernatant underwent IL-8 ELISA. *P<0.05, **P<0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NHEKs, normal human epidermal keratinocytes; RT-PCR, real-time PCR; siRNA, small interference RNA; TNF-α, tumor necrosis factor-α.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
IL-33 is expressed in the nuclei of epidermal keratinocytes in skin samples from patients with atopic dermatitis, lichen planus, and psoriasis. Formalin-fixed, paraffin-embedded skin samples from three patients with atopic dermatitis and five patients each with lichen planus and psoriasis were retrieved from the archives of the Department of Dermatology, Jichi Medical University. They were sliced into 4-μm thick sections, autoclaved for 10minutes in citrate buffer, and immunostained with anti-IL-33 antibody. Representative pictures are shown. (a) Normal control, (b) atopic dermatitis, (c) lichen planus, and (d) psoriasis. Bar=20μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions