Cockayne Syndrome Type A Protein Protects Primary Human Keratinocytes from Senescence  Sonia Cordisco, Lavinia Tinaburri, Massimo Teson, Donata Orioli, Romilda Cardin, Paolo Degan, Miria Stefanini, Giovanna Zambruno, Liliana Guerra, Elena Dellambra  Journal of Investigative Dermatology  Volume 139, Issue 1, Pages 38-50 (January 2019) DOI: 10.1016/j.jid.2018.06.181 Copyright © 2018 The Authors Terms and Conditions

Figure 1 CS-A primary keratinocyte cultures display premature senescence features. (a) Cell shape and colony organization of CS6PV and NHK1 keratinocytes are shown as representative of CS-A and normal cultures, respectively. Arrowheads indicate enlarged and flattened cells in CS-A culture (scale bar = 300 μm). (b–d) Colony number and clonal type of CS-A (CS15PV, CS6PV, and CS16PV) and NHK (NHK1 and NHK2) keratinocytes at the first culture passage. (b) CFE of CS6PV and NHK1 cultures are shown as representative images of CS-A and normal keratinocytes, respectively. (c) The reported CFE values represent the number of colonies expressed as percentages of the number of seeded cells. (d) The reported paraclone levels in CS-A and NHK cultures represent the number of aborted colonies expressed as percentages of the total colony number. Significance is calculated by comparing CS-A vs. NHK1 (*P < 0.05, **P < 0.01, ***P < 0.001, ANOVA with Bonferroni post hoc analysis) and CS-A vs. NHK2 (#P < 0.05, ##P < 0.01, ANOVA with Bonferroni post hoc analysis). (e) Cellular senescence analysis by the SA-β-gal assay on NHK and CS cultures. The reported values represent the percentage of positive cells. In the inset, CS6PV and NHK1 cultures are shown as representative images of CS-A and normal keratinocytes, respectively (bars = 300 μm). Significance is calculated by comparing CS-A vs. NHK1 (**P < 0.01, ***P < 0.001, ANOVA with Bonferroni post hoc analysis) and CS-A vs. NHK2 (##P < 0.01, ###P < 0.001, ANOVA with Bonferroni post hoc analysis). (f) p16 and p63 expression assessed by western blot using cell extracts from CS-A (CS15PV, CS6PV, and CS16PV) and NHK (NHK1 and NHK2) cultures. Densitometric signals are normalized to the GAPDH amount and values are expressed as fold changes (*P < 0.05, **P < 0.01, ANOVA with Bonferroni post hoc analysis). (g) ROS levels are expressed as DCFH fluorescence levels/cell. Significance is calculated by comparing CS-A vs. NHK1 (*P < 0.05, ***P < 0.001, ANOVA with Bonferroni post hoc analysis) and CS-A vs. NHK2 (#P < 0.05, ##P < 0.01, ANOVA with Bonferroni post hoc analysis). (h) Levels of 8-OH-dG residues in genomic DNA from CS-A (CS15PV, CS6PV, and CS16PV) and NHK (NHK1 and NHK2) measured by HPLC/electrochemical detection methodology. The levels of 8-OH-dG are expressed as the number of 8-OH-dG adducts per 106 deoxyguanosine (dG) bases. H2O2-treated cultures are used as positive controls. Significance is calculated by comparing CS-A vs. NHK1 (*P < 0.05, **P < 0.01, ***P < 0.001, ANOVA with Bonferroni post hoc analysis) and CS-A vs. NHK2 (#P < 0.05, ##P < 0.01, ###P < 0.001, ANOVA with Bonferroni post hoc analysis). 8-OH-dG, 8-OH-hydroxy-2′-deoxyguanosine; ANOVA, analysis of variance; CFE, colony-forming efficiency; CS, Cockayne syndrome; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NHK, normal human keratinocyte; ROS, reactive oxygen species; SA-β-gal, senescence-associated beta-galactosidase. Journal of Investigative Dermatology 2019 139, 38-50DOI: (10.1016/j.jid.2018.06.181) Copyright © 2018 The Authors Terms and Conditions

Figure 2 The transduced chimeric EGFP-CSAwt protein shows proper nuclear localization and restores normal response to UV irradiation and oxidative DNA damage levels. (a) Localization of EGFP alone or EGFP-CSAwt by fluorescence microscopy in CS6PV-V and CS6PV-CSAwt keratinocytes as representative of the transduced keratinocytes from the three patients with CS-A (scale bar = 100 μm). (b) Cell survival after UV irradiation of NHK (black symbols), CS-V (white symbols), and CS-CSAwt (gray symbols) cultures by the colony-forming assay. The numbers of colonies in irradiated samples are expressed as percentages of those in unirradiated samples. Each experiment was performed in triplicate and bars indicate the SD. (c) Capacity to recover RNA synthesis (RRS) 24 hours after UV irradiation by autoradiography in CS6PV-V and CS6PV-CSAwt keratinocytes as representative of the transduced keratinocytes from the three patients with CS-A. RRS is expressed as the mean number of autoradiographic grains/nucleus. The reported values are the mean of three independent experiments and bars indicate the SD. Significance is calculated by comparing CS-A vs. NHKs (**P < 0.01, Student’s t-test). (d) Levels of 8-OH-dG residues in genomic DNA from NHK, CS-V, and CS-CSAwt cultures in basal condition and immediately after UV irradiation (20 J/m2) by HPLC-electrochemical detection. The levels of 8-OH-dG are expressed as the number of 8-OH-dG adducts per 106 deoxyguanosine (dG) bases (8-OH-dG/106dG). Means of three independent experiments are reported and bars indicate the SD. Significance is calculated by comparing CS-A vs. NHKs (**P < 0.01, Student's t-test). (e) 8-OH-dG levels in NHK (black circles), CS-V (white squares), and CS-CSAwt (gray squares) cultures at different repair times are expressed as percentage of the levels immediately after UV irradiation. The reported values are the mean of three independent experiments each done in triplicate, and bars indicate the SD. 8-OH-dG, 8-OH-hydroxy-2′-deoxyguanosine; CS, Cockayne syndrome; EGFP, enhanced green fluorescent protein; NHK, normal human keratinocyte; SD, standard deviation. Journal of Investigative Dermatology 2019 139, 38-50DOI: (10.1016/j.jid.2018.06.181) Copyright © 2018 The Authors Terms and Conditions

Figure 3 The chimeric EGFP-CSAwt protein restores proper keratinocyte clonogenic ability. (a) Images of CS6PV-EGFP and CS6PV-CSAwt colonies are shown as representatives of the three analyzed CS-A keratinocyte strains (scale bar = 300 μm). (b–d) Colony number and clonal type are analyzed comparing CFE of CS-V (CS15PV-V, CS6PV-V, and CS16PV-V) and CS-CSAwt (CS15PV-CSAwt, CS6PV-CSAwt, and CS16PV-CSAwt) cultures. (b) The CFE assay of CS6PV-EGFP and CS6PV-CSAwt cultures is shown as representative of analyzed CS-A keratinocyte strains. (c) CFE values of CS-V and CS-CSAwt are expressed as the ratio of the total number of colonies to the number of inoculated cells (*P < 0.05, **P < 0.01, Student's t-test). (d) The paraclone percentage of CS-V and CS-CSAwt is expressed as the ratio of aborted colonies to the total number of colonies (*P < 0.05, **P < 0.01, Student's t-test). (e) Cellular senescence analysis by the SA-β-gal assay on CS-V and CS-CSAwt cultures. The reported values represent the mean percentage of positive cells (*P < 0.05, **P < 0.01, Student's t-test). Bars indicate the standard deviation. (f) p16 and p63 expression assessed by western blot using cell extracts of NHK, CS-V, and CS-CSAwt cells. Densitometric signals are normalized to the GAPDH amount and values are expressed as fold changes (*P < 0.05, Student's t-test). CFE, colony-forming efficiency; CS, Cockayne syndrome; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NHK, normal human keratinocyte; SA-β-gal, senescence-associated beta-galactosidase. Journal of Investigative Dermatology 2019 139, 38-50DOI: (10.1016/j.jid.2018.06.181) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Gene correction restores basal NF-κB activity and secretion of SASP mediators. (a, b) pAKT/AKT and IκBα expression assessed by western blot in cell extracts of NHK, CS-V, and CS-CSAwt cells. Densitometric signals are normalized to the GAPDH amount and values are expressed as fold changes (CS-CSAwt vs. CS-V, #P < 0.05, ##P < 0.01, Student's t-test). (c) NF-κB activity assessed by the ELISA-based assay in cell extracts of NHK, CS-V, and CS-CSAwt cells. Significance is calculated by comparing CS-V vs. NHK (*P < 0.05, **P < 0.01, ***P < 0.001, ANOVA with Bonferroni post hoc analysis) and CS-CSAwt vs. CS-V (#P < 0.05, ##P < 0.01, ###P < 0.001, ANOVA with Bonferroni post hoc analysis). (d) Expression of SASP factors, namely chemokines (CCL2/MCP-1, CCL5/RANTES, CXCL1/GRO-α, CXCL5/ENA 78, CXCL6/GCP2, CXCL8/IL-8, G-CSF), ILs (IL1-a, IL-4, IL-6, IL-6ST/SGP130, IL-10, IL-11, IL-16, IL-17), growth factors (BDNF, NT3, EGF, SCF, IGFBP2, VEGF D/FIGF, PLGF, TGFβ1, TNFRSF18, TNFα, and TNFβ), and MMPs/MMP inhibitors (MMP1, MMP3, MMP9, MMP10, MPP13, TIMP1, TIMP2), assayed by antibody arrays in supernatants of NHK, CS-V, and CS-CSAwt cultures. Signal intensity (S.I.) is normalized to the cell number. Significance is calculated by comparing CS-V vs. NHK (*P < 0.05, **P < 0.01, ***P < 0.001, ANOVA with Bonferroni post hoc analysis) and CS-CSAwt vs. CS-V (#P < 0.05, ##P < 0.01, ###P < 0.001, ANOVA with Bonferroni post hoc analysis). Akt, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MCP-1, monocyte chemotactic protein-1; MMP, metalloprotease; NHK, normal human keratinocyte; SASP, senescence-associated secretory phenotype; TGFβ1, transforming growth factor-β1; TIMP, tissue inhibitor of metalloproteinase; TNFα, tumor necrosis factor-α; VEGF, vascular endothelial growth factor. Journal of Investigative Dermatology 2019 139, 38-50DOI: (10.1016/j.jid.2018.06.181) Copyright © 2018 The Authors Terms and Conditions

Figure 5 Gene correction restores the cellular redox balance. (a) ROS levels are expressed as DCFH fluorescence levels/cell. Significance is calculated by comparing CS-CSAwt vs. CS-V (*P < 0.05, **P < 0.01, Student's t-test). (b, c) NOX1, MnSOD, and Catalase expression was evaluated by quantitative RT-PCR analysis on RNA extracted from NHK, CS-V, and CS-CSAwt cells. The RNA levels were normalized using the GUSB gene as housekeeping gene. Results were expressed as relative levels of RNA referred to a calibrator sample (NHK) that was chosen to represent 1x expression of this gene. Significance is calculated by comparing CS-V vs. NHK (*P < 0.05, **P < 0.01, ***P < 0.001, ANOVA with Bonferroni post hoc analysis) and CS-CSAwt vs. CS-V (#P < 0.05, ##P < 0.01, ###P < 0.001, ANOVA with Bonferroni post hoc analysis). (d) MnSOD and Catalase expression assessed by western blot in cell extracts of NHK, CS-V, and CS-CSAwt cells. Densitometric signals are normalized to the GAPDH amount and values are expressed as fold changes (CS-CSAwt vs. CS-V, **P < 0.01, Student's t-test). (e) The levels of 8-OH-dG of NAC-treated cultures are expressed as the number of 8-OH-dG adducts per 106 deoxyguanosine (dG) bases. Significance is calculated by comparing NAC-treated vs. untreated cells (**P < 0.01, Student's t-test). (f) pAKT/AKT, p16, and p63 expression assessed by western blot in cell extracts of NAC-treated and untreated cultures. Densitometric signals are normalized to the GAPDH amount and values are expressed as fold changes (CS+NAC vs. CS, *P < 0.05, Student's t-test). (g) NF-κB activity assessed by the ELISA-based assay in cell extracts of NAC-treated and untreated cultures. Significance is calculated by comparing NAC-treated vs. untreated cells (*P < 0.05, Student's t-test). (h) NOX1 was evaluated by quantitative RT-PCR analysis on RNA extracted from NAC-treated and untreated cultures. The RNA levels were normalized using the GUSB gene as housekeeping gene. Results were expressed as relative levels of RNA calibrator sample (untreated CS15PV). Significance is calculated by comparing NAC-treated vs. untreated cells (*P < 0.05, Student's t-test, **P < 0.01, Student's t-test). (i) CFE assay was assessed on NAC-treated and untreated cultures. The paraclone percentage is expressed as the ratio of aborted colonies to the total number of colonies (*P < 0.05, **P < 0.01, Student's t-test). 8-OH-dG, 8-OH-hydroxy-2′-deoxyguanosine; Akt, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; ANOVA, analysis of variance; CFE, colony-forming efficiency; CS, Cockayne syndrome; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NAC, N-acetylcysteine; NHK, normal human keratinocyte; ROS, reactive oxygen species. Journal of Investigative Dermatology 2019 139, 38-50DOI: (10.1016/j.jid.2018.06.181) Copyright © 2018 The Authors Terms and Conditions