Role of Matrix Metalloproteinases in HaCaT Keratinocytes Apoptosis Induced by Loxosceles Venom Sphingomyelinase D  Danielle Paixão-Cavalcante, Carmen W. van den Berg, Matheus de Freitas Fernandes-Pedrosa, Rute M. Gonçalves de Andrade, Denise V. Tambourgi  Journal of Investigative Dermatology  Volume 126, Issue 1, Pages 61-68 (January 2006) DOI: 10.1038/sj.jid.5700049 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Effect of L. intermedia venom and rP2 treatment on human keratinocytes cell viability. HaCaT cell cultures (1 × 105 cells/ml) were incubated with increasing concentrations of Loxosceles venom (·) or rP2 (○), and after 3 days of treatment, cell viability was analyzed by the Alamar Blue assay. Results are representative of two independent experiments and are expressed as the mean of triplicates ±standard deviation. The asterisks indicate values statistically different (P<0.05) from the control. Journal of Investigative Dermatology 2006 126, 61-68DOI: (10.1038/sj.jid.5700049) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Loxosceles venom SMaseD induces the expression of gelatinases in HaCaT cell line. Zymography analysis of HaCaT cell culture supernatants after 3 days of treatment with medium (C), 15μg/ml of venom (V), or rP2 (a). The supernatants of HaCaT cell cultures treated with 15μg/ml of venom (V) or rP2 or medium (C), in the presence or absence of tetracycline (50μg/ml) for 3 days, were run on 12.5% SDS-PAGE gel under nonreducing conditions and Western blotted using MoAbs against human MMP-2 (b) or anti-MMP-9 (c). Journal of Investigative Dermatology 2006 126, 61-68DOI: (10.1038/sj.jid.5700049) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of tetracycline, doxycycline, and minocycline on loss of cell viability of human keratinocytes induced by L. intermedia venom or rP2. HaCaT cells (1 × 105 cells/ml) were cultivated in 24-well plates in DMEM without FBS. At day 0, cells were treated with 15μg/ml of venom (a) or rP2 (b) and simultaneously incubated with different concentrations of tetracycline (▴), doxycycline (▵), or minocycline (○). Three days of treatment, the cell viability was analyzed by the Alamar Blue assay. Results are representative of two independent experiments and are expressed as the mean of triplicates ±standard deviation. The asterisks indicate values statistically different (P<0.05) from the control. Journal of Investigative Dermatology 2006 126, 61-68DOI: (10.1038/sj.jid.5700049) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effect of L. intermedia venom and SMaseD on the expression of keratinocyte cell surface antigens. HaCaT cells (107 cells/ml) were treated with buffer (C), Loxosceles venom (V), or rP2 (15μg/ml) for 2hours at 37°C in the presence or absence of tetracycline (50μg/ml) or EDTA (10mm). Binding of the SMaseD (a), using anti-F35 polyclonal antibody, and expression of MCP (b), β2-microglobulin (c), and EGFR (d), using specific MoAbs, were analyzed by flow cytometry. Results are representative of three different experiments expressed as the mean of duplicates±standard deviation. Journal of Investigative Dermatology 2006 126, 61-68DOI: (10.1038/sj.jid.5700049) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Binding of Annexin V and PI to HaCaT treated with L. intermedia venom and rP2. HaCaT cell cultures, in DMEM without FBS, were treated with 15μg/ml of venom or rP2 for 24hours at 37°C. As control, the cells were incubated with buffer. After incubation, cells were detached, washed in cold PBS, and incubated with Annexin V-FITC and PI as recommended by the manufacturer (R&D Systems Inc.) and analyzed by flow cytometry. Journal of Investigative Dermatology 2006 126, 61-68DOI: (10.1038/sj.jid.5700049) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 DNA gel eletrophoresis. HaCaT cells, cultured in DMEM without FBS, were treated with 15μg/ml of venom (V) or rP2 for 24hours at 37°C in the presence (+) or absence of tetracycline (50μg/ml). Cells incubated with PBS (C) and hydrogen peroxide (H2O2) (100μm) were used as negative and positive controls, respectively. After incubation, cells were detached, washed in cold PBS, and the DNA was isolated using Trizol reagent and analyzed by agarose gel electrophoresis. Journal of Investigative Dermatology 2006 126, 61-68DOI: (10.1038/sj.jid.5700049) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions