1. NADPH Oxidase-1 Plays a Key Role in Keratinocyte Responses to UV Radiation and UVB-Induced Skin Carcinogenesis 
Houssam Raad, Martin Serrano-Sanchez, Ghida Harfouche, Walid Mahfouf, Doriane Bortolotto, Vanessa Bergeron, Zeinab Kasraian, Lea Dousset, Mohsen Hosseini, Alain Taieb, Hamid Reza Rezvani 
Journal of Investigative Dermatology 
Volume 137, Issue 6, Pages (June 2017)
DOI: /j.jid
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2. Figure 1
Biphasic effect of UVB irradiation on NOX activity. (a) NOX activity was measured at indicated time points. The results are expressed as mean ± standard deviation of three independent experiments. ∗P < 0.05 and ∗∗P < 0.01 versus cells before irradiation. (b) Total protein extracts were assessed for the presence of different subunits of NOX1, NOX2, and NOX4 complexes by Western blot. GAPDH was used as a loading control. (c–e) The cell lysates were immunoprecipitated with (c) anti-p22phox, (d) anti-NOX1, and (e) anti-NOX2 and analyzed by Western blot for co-immunoprecipitated levels of different components of NOX complexes. Part of the cell lysate was used as an input to show the endogenous level of each protein, and immunoprecipitation with IgG was used as negative control. Ctrl, control; GADPH, glyceraldehyde-3-phosphate dehydrogenase; h, hour; HC, heavy chain of immunoprecipitating antibody; LC, light chain of immunoprecipitating antibody; nIr, nonirradiated; NOX, nicotinamide adenine dinucleotide phosphate oxidase; sh, short hairpin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
Treatment of keratinocytes before exposure to UVB with InhNOX1 or GKT leads to increased removal rate of UVB-induced DNA damage and decreased apoptosis. (a) NOX activity and (b) co-immunoprecipitated levels of different components of NOX1 complexes were assessed. The percentages of (c) 6-4PPs and (d) CPD remaining at various time points after irradiation was evaluated by comparison with the initial levels. (e) The quantity of CPD was assessed by immunodot blot. SYBR green was used as a loading control. (f) The relative activities of caspase-3, -8, and -9 were measured. The activity of each caspase in the scrambled-treated cells was arbitrarily set to 100. (g) Total protein extracts were assessed for cell-cycle regulators and activation of caspases. β-actin was used as a loading control. The results are expressed as the mean ± standard deviation of three independent experiments. ∗P < 0.05 and ∗∗P < 0.01 for treated cells at the indicated time points versus corresponding scrambled-treated cells. 6-4PP, 6-4 photoproducts; CPD, cyclobutane pyrimidine dimer; h, hour; HC, heavy chain of immunoprecipitating antibody; InhNOX, peptide inhibitor of NOX1; IP, immunoprecipitate; Ir, irradiated; LC, light chain of immunoprecipitating antibody; NAPDH, nicotinamide adenine dinucleotide phosphate; nIr, nonirradiated; NOX, nicotinamide adenine dinucleotide phosphate oxidase; p-, phosphorylated.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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4. Figure 3
Treatment of keratinocytes with InhNOX1 or GKT hour after exposure to UVB results in decreased removal rate of UVB-induced DNA damage and apoptosis. (a) NOX activity and the levels of (b) 6-4PPs and (c) CPDs were assessed at various time points after irradiation by comparison with the initial levels. (d) The quantity of CPD was evaluated by immunodot blot. SYBR green was used as a loading control. (e) The relative activities of caspase-3, -8, and -9 were measured at 20 hours after irradiation. The activity of each caspase in the scrambled-treated cells was arbitrarily set to 100. (f) Total protein extracts were assessed for cell cycle regulators and activation of caspases by Western blot analysis. β-actin was used as a loading control. Results are expressed as the mean ± standard deviation of three independent experiments. ∗P < 0.05 and ∗∗P < 0.01 for treated cells versus scrambled-treated cells at corresponding time point. 6-4PP, 6-4 photoproducts; CPD, cyclobutane pyrimidine dimer; h, hour; InhNOX, peptide inhibitor of NOX1; Ir, irradiated; NAPDH, nicotinamide adenine dinucleotide phosphate; nIr, nonirradiated; p-, phosphorylated.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
NOX1 inhibition increases removal rate of UVB-induced DNA damage and reduces photocarcinogenesis in XPC-proficient and -deficient mice. (a) One-month-old Xpc+/+ mice were treated with GKT or InhNOX1 for 4 months. Histopathology of epidermis was evaluated. Scale bars = 50 μm. (b–e) Xpc+/+ and Xpc–/– mice were subjected to chronic UVB irradiation. (b) Variable numbers of tumors of variable size that are mostly ulcerated are visible on the back of mice. The (c) number and (d) volume of tumors per mouse were assessed. (e) The tumor volume distribution was evaluated at week 24. (f) NOX activity was evaluated in nonirradiated (nIr) mouse skin, non-tumor bearing and tumors. (g) The CPD level was evaluated, quantified densitometrically, and normalized to SYBR green intensity. (h) Relative activity of caspase-3 was measured. Results are expressed as the mean ± standard deviation. n = 6 mice per group in a, n = 20 in b–e, n = 8 in f and h, and n = 15 in g. CPD, cyclobutane pyrimidine dimer; h, hours; H&E, hematoxylin and eosin; InhNOX, peptide inhibitor of NOX1; Ir, irradiated; K, keratin; min, minutes; NAPDH, nicotinamide adenine dinucleotide phosphate; NOX, nicotinamide adenine dinucleotide phosphate oxidase; XPC, xeroderma pigmentosum type C.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
NOX1 inhibition slows down the growth of UVB-induced skin tumors in XPC-proficient and -deficient mice. (a–d) Xpc+/+ and Xpc–/– mice were subjected to chronic UVB irradiation. The mice received scrambled peptide or InhNOX1 topically 10 minutes before each irradiation. Antioxidant (a) Trolox capacity, (b) catalase, (c) CuZnSOD, and (d) MnSOD activities in mouse skin were evaluated. ∗∗∗P < versus nonirradiated XPC-proficient mice conditions; °P < 0.05 tumors versus non-tumor bearing skin, •P < 0.05 versus nonirradiated XPC-deficient mice. (e–g) Effects of InhNOX1 treatment on the growth of UVB-induced skin tumors. (e) Drawing showing experimental protocol design. The effects of InhNOX1 treatment on the (f) number and (g) volume of tumors per mouse were assessed. ∗P < 0.5 versus scrambled-treated counterpart controls at corresponding time point. Magenta and blue-violet ∗P < 0.05 for InhNOX1-treated mice at indicated time versus starting point. (a–d) n = 20 and (e–g) 15 mice per group. InhNOX, O-1 peptide; h, hour; nIr, nonirradiated; XPC, xeroderma pigmentosum type C.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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