DDB2, XPC and p53 follow same induction/translocation pattern after UV exposure in NHEK and HCEC.

<p>Quantification and distribution of DDB2, XPC and p53 after UV exposure in NHEK and HCEC was assessed by Western immunoblot. (A) Representative image of Western immunoblots for DDB2 and XPC analysed from chromatin bound (CB) and for DDB2, XPC and p53 from nuclear soluble (NS) protein extracts of NHEK and HCEC cultures harvested before (-) and at different time points following a 400 J/m² of UVB irradiation. (B) Quantitative analysis of Western blots shows a rapid increase of DDB2 on the CB fraction and a (E) rapid decrease in NS post-UV irradiation (unirradiated control vs 0h post-UV) in both NHEK and HCEC. This is followed by a partial or complete restoration of DDB2 level to the unirradiated control level in NS and CB fractions, respectively. (C) and (F) For XPC, the protein level varies to a lesser extent post-irradiation and was considered non-significant in both NHEK and HCEC. No significant difference in DDB2 and XPC levels modulation post-irradiation is observed between NHEK and HCEC. (G) For p53, the protein levels increase drastically 1h post-irradiation for at least 4h. Each western is quantified independently and is not compared with each other. The protein levels of DDB2, XPC or p53 post-irradiation are calculated as the percentage of variation in relation to the unirradiated level for each protein. A value of 1 is attributed to the NoUV for each protein and the post-irradiation levels are related to the NoUV. Data information: Data presented as means ± SEM. No significant difference was raised; Student’s <b><i>t</i></b>-test. Experiment was repeated on 5 NHEK (N = 5) and 4 HCEC (N = 4) in duplicate (n = 2).</p

Map of Guinea-Bissau.

<p>Map of Guinea-Bissau.</p

NHEK are more sensitive to UV-induced cell death when compared to HCEC.

<p>Apoptosis and necrosis is assessed by Annexin V/FITC and PI assay in UVB-irradiated NHEK and HCEC with doses ranging from 1,000 J/m² to 5,000 J/m² of UVB at 16h post-irradiation. Dotted, dashed and plain bars represent necrosis, apoptosis and live cells, respectively. Data information: Data presented as means ± SEM. Percentage of necrotic, apoptotic and live cells are significantly different (p<0.05) between NHEK and HCEC irradiated at 5,000 J/m² UVB, according to the Student’s <b><i>t</i></b>-test. Experiment was repeated on 3 NHEK cultures (N = 3) and 3 HCEC cultures (N = 3) in triplicate (n = 3).</p

DDB2, XPC and p53 protein levels are higher in HCEC than NHEK.

<p>Representative Western immunoblots for DDB2 (A), XPC (B) and p53 (C) analysed in the chromatin bound (CB) and nuclear soluble (NS) protein extracts from NHEK and HCEC. Coomassie blue staining was used as a loading control. (A) The quantitative analysis of Western blots shows DDB2 protein levels higher in HCEC when compared to NHEK in both CB and NS fractions. (B) As for XPC, protein level is significantly higher in HCEC than NHEK only in the NS faction. (C) p53 level is significantly higher in NS fraction of HCEC when compared to NHEK. Relative HCEC protein levels are related to their NHEK protein levels counterparts. Data information: Data presented as means ± SEM. *<b><i>P</i></b> < 0.05, *<b><i>P</i></b> < 0.01; Student’s <b><i>t</i></b>-test. (A to F) Experiment was repeated on 5 NHEK (N = 5) and 9 HCEC (N = 9) in triplicate (n = 3).</p

NER damage recognition proteins DDB2 and XPC are longer-lived in HCEC than NHEK.

<p>DDB2 and XPC protein half-lives in NHEK and HCEC were assessed by cycloheximide chasse assay. (A) Representative image of Western immunoblots of DDB2 and XPC analysed from chromatin bound (CB) and nuclear soluble (NS) fractions of NHEK and HCEC cultures without (-) and with added cycloheximide to the medium for different times to inhibit <i>de novo</i> protein synthesis. (B), (C), (D), and (E) Quantitative analysis of Western blots shows a decrease of CB and NS fractions of DDB2 and XPC over time post cycloheximide exposure in both NHEK and HCEC. However, the decrease is faster in NHEK for both CB and NS fractions when compared to HCEC, indicating a greater half-lives of DDB2 and XPC proteins in HCEC. Percentages of protein remaining are calculated by relating the intensity of signals from cells exposed to cycloheximide to the signals from their unexposed counterparts. Data information: Data presented as means ± SEM. *<b><i>P</i></b> < 0.05, **<b><i>P</i></b> < 0.01, ***<b><i>P</i></b> <0.001; Student’s <b><i>t</i></b>-test. Experiment was repeated on 4 NHEK (N = 4) and 4 HCEC (N = 4) in duplicate (n = 2).</p

CPD repair, but not 6-4PP, is more efficiently in HCECN than NHEK.

<p>UVB-induced CPD and 6-4PP repair kinetics in NHEK and HCEC by DNA slot-blot. (A) and (B) Representative images of DNA slot-blots. DNA extracts from NHEK and HCEC cultures (<i>in vitro</i>) along with corneal epithelium (HCEC <i>ex vivo</i>) harvested before (-) and at different time points following a 400 J/m² of UVB irradiation were blotted. UV-induced DNA damage was revealed with specific antibodies; (A) CPD and (B) 6-4PP. (C) and (D) Quantitative analysis of DNA slot-blots. Percentages of DNA damage remaining are calculated by relating the intensity of signals in the DNA of irradiated cells to the intensity of their unirradiated counterparts. The analysis shows that repair of CPD, but not 6-4PP, is significantly faster in HCEC when compared to NHEK. Data information: (C and D) Data presented as means ± SEM. (C) <b><i>P</i></b>-values were calculated between NHEK <i>in vitro</i> and HCEC <i>in vitro</i> only. *<b><i>P</i></b> < 0.05, **<b><i>P</i></b> < 0.01; Student’s <b><i>t</i></b>-test. (A and C) Experiment was repeated on 4 NHEK <i>in vitro</i> (N = 4), 7 HCEC <i>in vitro</i> (N = 7) and 9 HCEC <i>ex vivo</i> (N = 9) in duplicate (n = 2). (B and D) Experiment was repeated on 4 NHEK <i>in vitro</i> (N = 4) and 6 HCEC <i>in vitro</i> (N = 6) once (n = 1).</p

More CPD are found in corneal explants than in skin explants following a same dose of UV-irradiation but similar levels of CPD are detected in cultured NHEK and HCEC.

<p>UVB-induced CPD formation in skin and cornea was assessed in human skin biopsies, eyes and cultured NHEK and HCEC irradiated with 2,000 J/m² of UVB. CPD were detected with an anti-CPD antibody (green) and DNA was counterstained with DAPI (red). (A) Immunohistofluorescence of UVB-induced CPD in skin and cornea shows an important induction of CPD in both epidermis and corneal epithelium. (B) Quantitative analysis of immunohistofluorescence. Relative CPD/DNA ratio from the epidermis and the corneal epithelium indicates that 3.4 times more CPD are found in the corneal epithelium from explants than in the epidermis from skin explants. (C) Immunocytofluorescence of UVB-induced CPD in cultured HCEC and NHEK. (D) Quantitative analysis of immunocytofluorescence. Relative CPD/DNA ratio from HCEC and NHEK show there is not difference in CPD induction between both cell types. Data information: (A, C) Scale bar, 50μm. (B, D) Data presented as means ± SEM. ***<b><i>P</i></b> < 0.001; Student’s <b><i>t</i></b>-test. (A, B) Experiment was repeated on 5 skins (N = 5) and 4 corneas (N = 4) in duplicate (n = 2). (C, D) Experiment was repeated on 3 strains of NHEK and HCEC (N = 3).</p

Comparison of average probabilities of death among H1N1 ICU patients based on APACHE II severity of disease classification system.

<p>het, heterozygous; hom, homozygous.</p

KIR3DL1/S1 allele frequencies of ICU patients and St. Theresa aboriginals.

<p>AF, allele frequency; Ab, Aboriginal; NAb, Non-aboriginal.</p><p>STh, St. Theresa aboriginals; C, H1N1 confirmed cases.</p>1<p>Odds Ratio:3.031(CI95%:1.199–7.663);</p>2<p>Odds Ratio:4.556(CI95%:1.167–17.779).</p

Amplification and sequencing primers for KIR3DL1/S1 typing.

a<p>Does not amplify 3DL1*059, 3DL1*060, 3DL1*061.</p>b<p>3DL1 only.</p>c<p>Touchdown PCR temperature range.</p