1. Hypo-osmotic Stress Drives IL-33 Production in Human Keratinocytes—An Epidermal Homeostatic Response 
Wojciech Pietka, Denis Khnykin, Vibeke Bertelsen, Astrid Haaskjold Lossius, Tor Espen Stav-Noraas, Johanna Hol Fosse, Hilde Kanli Galtung, Guttorm Haraldsen, Olav Sundnes 
Journal of Investigative Dermatology 
Volume 139, Issue 1, Pages (January 2019)
DOI: /j.jid
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2. Figure 1
Hypo-osmotic stress induces IL-33 in primary human keratinocytes. (a, b) Immunofluorescent stainings of human skin tissue for pSTAT1 (green) and nuclear marker Topro-3 (blue). Dotted line marks dermoepidermal junction. (c–l) Keratinocytes exposed to isoosmotic, hypo-osmotic, or hyperosmotic medium and are examined by (c, d) immunofluorescence for IL-33 (red) and keratin 14 (green), (e) ELISA of cell lysates and Western blot, and (f) real-time PCR. (g) IL-33 induction verified by siRNA knockdown and Western blot analysis. (h–l) mRNA expression of various proinflammatory mediators in hypo-osmotic, isosmotic, and hyperosmotic conditions examined by real-time PCR. Data are shown as mean ± standard deviation of three replicates. ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗∗P ≤ (increase vs. isoosmotic); #P < 0.05 (decrease vs. isoosmotic). Scale bar = 50 μm. ns, non-significant. siRNA, small interfering RNA.
Journal of Investigative Dermatology  , 81-90DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

3. Figure 2
Primary keratinocytes show an atypical morphological response to hypo-osmotic shock. (a) Phase-contrast microscopy images of keratinocytes that were left exposed for 15 minutes in isoosmotic or hypo-osmotic medium. (b, c) IL-33 ELISA data of keratinocytes (b) exposed to hypo-osmotic medium before returning to isoosmotic medium or vice versa or (c) exposed for 6 hours to medium diluted with 50% water, or 50% isoosmotic solutions of phosphate buffered saline, potassium gluconate, or sodium gluconate. ELISA data are shown as mean ± standard deviation of three replicates. ∗∗∗∗P ≤ (increase vs. isoosmotic). (d) Western blot of IL-33 and tubulin after 6 hours of isoosmotic medium, hypo-osmotic medium, isosmotic urea solution, or isoosmotic sorbitol solution. (e) Cell morphology after 15 minutes of isoosmotic urea or sorbitol solution exposure. Scale bar = 50 μm.
Journal of Investigative Dermatology  , 81-90DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

4. Figure 3
Stress- or IFN-γ–induced IL-33 both depend on EGFR signaling but have different upstream mediators. (a) Keratinocytes were stimulated in normal growth medium with recombinant EGF or exposed to hypo-osmotic medium with or without pre-incubation with EGFR tyrosine kinase inhibitor (AG1478) or anti-EGFR antibody (cetuximab) for 6 hours. (b) Keratinocytes were left in basal medium without growth factors 90 minutes before stimulations and subsequently were exposed to hypo-osmotic medium and/or different concentrations of amphiregulin for 6 hours. (c–h) Immunofluorescence of (c–e) IL-33 (grayscale) or (f–h) combination of IL-33 (red) and Ki67 (green) in keratinocytes that were either untreated, exposed to IFN-γ, or to hypo-osmotic stress. (i) Western blot of keratinocytes stimulated with either IFN-γ or hypo-osmotic medium with different concentrations of pan-JAK inhibitor I. Scale bar = 50 μm.
Journal of Investigative Dermatology  , 81-90DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

5. Figure 4
Calcium-dependent processes drive IL-33 production and modulate the general proinflammatory response. (a) IL-33 ELISA of keratinocyte lysates treated with calcium chelator BAPTA-AM before stimulation. Data are shown as mean ± standard deviation of three replicates. ∗∗∗∗P ≤ (b) Confluent keratinocytes switched to calcium-free medium and stimulated with IFN-γ or hypo-osmotic solution, adding either 0.07 mmol/L CaCl2 (low calcium), EGTA, BAPTA-AM chelator, or 1.2 mmol/L CaCl2 (high calcium). (c) Real-time PCR data of pro-inflammatory mediators from keratinocytes exposed to stressors with or without BAPTA-AM. Data represent fractions of maximum mRNA levels set to 100% within the four conditions. (d) Selected transcript levels from IL-33 siRNA-treated keratinocytes were analyzed by real-time PCR. Data show mean fold change ± standard deviation of three replicates. ∗∗∗P ≤ ns, nonsignificant; siRNA, small interfering RNA.
Journal of Investigative Dermatology  , 81-90DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

6. Figure 5
Hypo-osmotic stress is a relevant tissue signal. (a–d) Normal human skin tissue was either (a) immediately fixed in formalin or cultured ex vivo for 24 hours in (b) normal medium, (c) hypo-osmotic medium, or (d) with addition of IFN-γ with subsequent formalin fixation. Dotted line marks dermoepidermal junction. Sections of formalin-fixed paraffin-embedded tissue were stained with immunofluorescent technique for IL-33 (red), p-STAT1 (green), and nuclear stain with Hoechst (blue). (e–l) Single-color images of the same stainings are shown for (e–h) IL-33 (grayscale) and (i–l) pSTAT (grayscale). Scale bar = 50 μm.
Journal of Investigative Dermatology  , 81-90DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions