Immunoprotective UVA (320–400 nm) Irradiation Upregulates Heme Oxygenase-1 in the Dermis and Epidermis of Hairless Mouse Skin Munif Allanson, Vivienne.

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Immunoprotective UVA (320–400 nm) Irradiation Upregulates Heme Oxygenase-1 in the Dermis and Epidermis of Hairless Mouse Skin Munif Allanson, Vivienne E. Reeve Journal of Investigative Dermatology Volume 122, Issue 4, Pages 1030-1036 (April 2004) DOI: 10.1111/j.0022-202X.2004.22421.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 RT-PCR detection of HO-1 and HO-2 mRNAs in the skin of hairless mouse at various time points. Transcripts encoding HO-1 and HO-2 were separated on 1.2% agarose gel. Gel-1, unirradiated control skin (mouse a and b); Gel-2, UVB-irradiated skin; Gel-3, UVA-irradiated skin. HO-1 and HO-2 amplifications as follows: right to left, HO-1 (0, 1.5, 3, 6, 8, 12, and 24 h post-UV), DNA marker (GeneRuler 1kb DNA ladder Cat No. # SM0311, Fermentas Life Science, Brisbane, Australia), and HO-2 (0, 1.5, 3, 6, 8, 12, and 24 h post-UV). HO-2 mRNA level was used as housekeeping gene. HO-1 mRNA was expressed immediately and persisted 24 h post-UVA (Gel-3), but not in control (Gel-1) or UVB-irradiated skin (Gel-2). In contrast, HO-2 mRNA was always expressed at all time points in Gel-1, Gel-2, and Gel-3 Journal of Investigative Dermatology 2004 122, 1030-1036DOI: (10.1111/j.0022-202X.2004.22421.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Semi-quantitative image analysis of HO-1 mRNA expression in the skin at various time points using “Quantity One” program. HO-1 bands were normalized against HO-2 band, to obtain HO-1:HO-2 ratio. Time point “0” was taken immediately on completion of the irradiation (there was no detectable HO-1 mRNA in unirradiated skin). The peak expression of HO-1 mRNA was at 3 h post-UVA radiation Journal of Investigative Dermatology 2004 122, 1030-1036DOI: (10.1111/j.0022-202X.2004.22421.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Probes synthesis. Gel-1: from right to left, (M) DNA marker (GeneRuler 1kb DNA Ladder), (1) sense (Dig-labelled), (2) antisense (Dig-labelled), (3) sense and antisense together (Dig-labelled), (4) sense and antisense unlabelled, and (5) positive PCR control kit (Dig-labelled). Dig incorporation increases the molecular weights of the probes, which migrated slower in the gel compared with unlabelled probe (line 4). Gel-2: HO-1 mRNA of mouse liver at 90 min after LPS stimulation. The cDNA was used as a template for probes synthesis for HO-1. Note that the HO-1 mRNA band lies between 750 and 1000 bp Journal of Investigative Dermatology 2004 122, 1030-1036DOI: (10.1111/j.0022-202X.2004.22421.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 In situ hybridization. HO-1 mRNA expression at 3 h post-UVA showed a strong signal with the antisense probe (a) in cells of both the epidermis and dermis, but was absent with the unlabelled antisense probe (b) or the control sense probe or in unirradiated skin (not shown). In the epidermis, HO-1 mRNA expression can be seen in or near the basal region, and along the hair follicle lining. In the dermis, HO-1 mRNA expression can be seen in fibroblast-like cells. Immunohistochemistry: HO-1 protein expression at 72 h post-UVA (c) can be seen strongly in the epidermis, including along the cells lining the hair follicle. In the dermis, HO-1 protein expression can be seen in fibroblast-like cells (arrows). HO-1 protein expression was absent in unirradiated skin (d) or negative staining controls (not shown). Scale bar=50 μm Journal of Investigative Dermatology 2004 122, 1030-1036DOI: (10.1111/j.0022-202X.2004.22421.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Heme oxygenase activity in skin microsomes at 72 h post-UVA was significantly (p<0.05) induced. Error bars=SEM Journal of Investigative Dermatology 2004 122, 1030-1036DOI: (10.1111/j.0022-202X.2004.22421.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Microsomal heme was significantly (p<0.05) reduced at 72 h post-UVA compared with unirradiated microsomes, but not in the mitochondria. Error bars=SEM Journal of Investigative Dermatology 2004 122, 1030-1036DOI: (10.1111/j.0022-202X.2004.22421.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Skin bilirubin was significantly (p<0.05) elevated at 72 h post-UVA in skin compared with unirradiated skin. Error bars=SEM Journal of Investigative Dermatology 2004 122, 1030-1036DOI: (10.1111/j.0022-202X.2004.22421.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions