1. Journal of Investigative Dermatology
Spironolactone Depletes the XPB Protein and Inhibits DNA Damage Responses in UVB-Irradiated Human Skin 
Michael G. Kemp, Smita Krishnamurthy, Michael N. Kent, David L. Schumacher, Priyanka Sharma, Katherine J.D.A. Excoffon, Jeffrey B. Travers 
Journal of Investigative Dermatology 
DOI: /j.jid
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2. Figure 1
Spironolactone depletes XPB protein and abrogates DNA damage responses in UVB-irradiated, telomerase-immortalized (N-TERT) keratinocytes in vitro. (a) Immunoblot analysis of XPB protein levels after treatment with the indicated concentration of SP (n = 2). Molecular weight markers and a cross-reacting band (∗) are indicated. (b) Time course analysis of XPB loss after SP treatment (n = 2). (c) Immunodot blot analysis of CPD removal from genomic DNA (n = 3). Blots were probed sequentially with anti-CPD and anti-DNA antibodies. (d) Confluent N-TERTs grown for 24 hours in basal medium lacking growth factors were treated as indicated and harvested 0.5 hours after exposure to 100 J/m2 UVB to enrich for chromatin-associated proteins (n = 2). (e) Cells were treated as in d except that cells were harvested 1 hour after UVB exposure, and cell lysates were analyzed for ATR/ATM kinase substrate protein phosphorylation on the indicated protein residue (n = 4). (f) Cell survival analysis 2 days after exposure to the indicated fluence of UVB (n = 3). All graphs show the average ± standard error of the mean. ∗P < CPD, cyclobutane pyrimidine dimer; hrs, hours; SP, spironolactone.
Journal of Investigative Dermatology DOI: ( /j.jid )
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3. Figure 2
Spironolactone negatively affects XPB protein levels and UVB DNA damage responses in primary adult human keratinocytes in vitro. (a) Normal, primary, nonimmortalized keratinocytes from three different skin donors (NHK1, 2, and 3) were treated for 2 hours with DMSO or 10 μmol/L SP, and cell lysates were analyzed by immunoblotting. (b) ATR/ATM substrate protein phosphorylation was analyzed as in Figure 1d. (c) Analysis of CPD removal in normal keratinocytes was performed as described in Figure 1c. (d) Cell survival 2 days after exposure to 100 J/m2 of UVB. All graphs show the average ± standard error of the mean. ∗P < CPD, cyclobutane pyrimidine dimer; SP, spironolactone.
Journal of Investigative Dermatology DOI: ( /j.jid )
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4. Figure 3
SP depletes the XPB protein and modulates DNA damage responses in UVB-irradiated human skin ex vivo. (a) Skin explant culture medium was supplemented on 3 sequential days with DMSO or 20 μmol/L SP or eplerenone (EP). Epidermal lysates were analyzed by immunoblotting (n = 5–9). (b) Skin biopsy samples were treated with increasing concentrations of SP as in a (n = 3). (c) Skin biopsy samples treated for 3 days with DMSO or SP were exposed to 700 J/m2 UVB. Genomic DNA was purified from the epidermis and analyzed by immunodot blotting (n = 4–6). (d) Epidermal lysates were prepared 1 hour after UVB irradiation and analyzed by immunoblotting (n = 5). (e) Skin biopsy samples were treated as in c, fixed in formalin, and stained with hematoxylin and eosin. Scale bars = 0.1 mm. Apoptotic “sunburn” cells (indicated by white arrows) counted (n = 6). All graphs show the average ± standard error of the mean. ∗P < hrs, hours; SP, spironolactone.
Journal of Investigative Dermatology DOI: ( /j.jid )
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5. Figure 4
SP metabolites and the mineralocorticoid receptor antagonist eplerenone do not affect XPB protein levels or the UVB DNA damage response. (a) N-TERTs were treated for 3 hours with DMSO or with 10 μmol/L spironolactone (SP), canrenone (Can), 7α-thiomethylspironolactone (TMS), or eplerenone (EP). Cell lysates were analyzed by immunoblotting (n = 3–5). (b) N-TERTs treated as in a were irradiated with 100 J/m2 UVB and harvested for analysis of CPD levels by immunodot blotting (n = 3). (c) Cells treated as in a were analyzed for cell survival (n = 3–6). (d) HaCaT cells were treated for 2 hours with DMSO or 300 nmol/L SP, Can, or EP before exposure to 100 J/m2 UVB. The number of 6-thioguanine–resistant clones (per million plated cells) was quantified 2 weeks later (n = 2–3). All graphs show the average ± standard error of the mean. ∗P < CPD, cyclobutene pyrimidine dimer; hrs, hours; N-TERT, telomerase-immortalized neonatal foreskin keratinocytes.
Journal of Investigative Dermatology DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions