1. HMGB1/RAGE Mediates UVB-Induced Secretory Inflammatory Response and Resistance to Apoptosis in Human Melanocytes 
Kun Zhang, Govindaraj Anumanthan, Suzanne Scheaffer, Lynn A. Cornelius 
Journal of Investigative Dermatology 
Volume 139, Issue 1, Pages (January 2019)
DOI: /j.jid
Copyright © 2018 The Authors Terms and Conditions

2. Figure 1
UVB induces a DNA damage response and a persistent gene signature, characteristic of senescence associated secretory phenotype in human primary melanocytes. (a) HEMn-LP cells were exposed to UVB (100 mJ/cm2) x 1. At varying time points after exposure, cells were collected and mRNA was extracted. Quantitative PCR determination of mRNA levels for gene expression of specific ISGs as indicated was performed. (b) HEMn-LP cells were treated as in (a), cells were collected, and western blot was performed to determine protein expression and activation of tyrosine kinases and transcription factors as indicated at varying time points. (c) HEMn-LP cells were treated as in (a). Twenty-four hours after UVB exposure, cell supernatants were collected. CCL1, CCL2, IL6, and IL8 were determined by ELISA. (d) HEMn-LP and HEMn-DP cell pellets and melanin quantifications were shown as μg/2.5 × 104 cells. (e) HEMn-LP and HEMn-DP cells were treated with UVB (100 mJ/cm2) every 3 days for total five times (intermittently exposed). Twenty-four hours after the last UVB exposure (14 days), cells were collected and mRNA was extracted. Quantitative PCR determination of gene expression for indicated genes was performed. AKT, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; ERK, extracellular signal-regulated kinase; STAT1, signal transducer and activator of transcription 1.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

3. Figure 2
UVB induced senescence and persistent senescence-associated secretory phenotype response results in relative resistance to further DNA damage and apoptosis. (a) HEMn-LP and HEMn-DP cells were either sham-exposed (left panel), exposed to single dose UVB (100 mJ/cm2) (middle panel), or intermittent UVB (100 mJ/cm2) (right panels) for 28 days, maintained in culture and passaged accordingly. Immunofluorescent staining was performed to identify nuclei DAPI (blue), H2A.X (s139) (green), and F-actin (Rhodamine-phalloidin-red) in HEMn-LP and HEMn-DP cells at 1 day after single exposure and at 28 days after intermittent exposure. (Bottom left) Cyclobutane pyrimidine dimer (CPD) formation was quantified by ELISA (Cell BioLab). At 1 day after single UVB exposure, CPD formation was similar in both HEMn-LP and HEMn-DP cells. At 28 days after intermittent exposure, the relative change in CPD formation was significantly decreased in HEMn-LP cells compared with HEMn-DP cells (*P < 0.05). (Bottom right) Quantitation of the H2A.X signal was performed with the ImageJ software (NIH). At 1 day after single UVB exposure H2A.X foci formation was similar in both HEMn-LP and HEMn-DP cells. At 28 days after intermittent exposure, HEMn-LP retained significantly higher levels of H2A.X foci compared with HEMn-DP (*P < 0.05) (scale bar = 10 μm). (b) HEMn-LP and HEMn-DP cells were either sham-exposed (top row) or exposed to UVB (middle row). UVB exposure was either single dose UVB (100 mJ/cm2) (1 day) or intermittent UVB (100 mJ/cm2 at 14 days) or intermittent UVB and maintained in culture for an additional 14 days (28 days). Cells were fixed and stained for expression of senescence-associated beta galactosidase (SA-β-gal). (Upper two panels) The corresponding cell morphology of UVB-exposed cells at each time point is shown by phase-contrast microscopy in the lower panel. Quantification based on SA-β-gal staining was performed by cell counts in triplicate and calculation of the percentage of positively stained cells (scale bar = 50 μm). (c) HEMn-LP and HEMn-DP cells were treated as in (b), cell lysates were prepared, and western blot was performed for protein expression of p21, p53, p16, STAT1 and pSTAT, MX1, AKT and pAKT, p-p38, and pErk 1/2. β-Tubulin was used as control. (d) After intermittent exposure, HEMn-LP and HEMn-DP cells were maintained for another 14 days in culture (28 days). Cells were then collected and mRNA was extracted. qPCR was performed for gene expression of genes that were differentially regulated at 14 days. (e) Intermittent UVB-treated HEMn-LP and HEMn-DP cells were treated with doxorubicin (1–10 μg/ml) for 2 days after last exposure. Cell viability was determined by the MTT assay. (f) Intermittent UVB-treated HEMn-LP cells were treated with high doses of UVB (300 and 600 mJ/cm2) 48 hours after the last UVB exposure. Twenty-four hours after high-dose exposure, cells were collected and stained with the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & PI. The apoptotic and dead cells were detected by FACS analysis (upper panel). The quantification data are shown in graphic form based on FACS data of three independent experiments. AKT, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; ERK, extracellular signal-regulated kinase; STAT1, signal transducer and activator of transcription 1.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

4. Figure 2
UVB induced senescence and persistent senescence-associated secretory phenotype response results in relative resistance to further DNA damage and apoptosis. (a) HEMn-LP and HEMn-DP cells were either sham-exposed (left panel), exposed to single dose UVB (100 mJ/cm2) (middle panel), or intermittent UVB (100 mJ/cm2) (right panels) for 28 days, maintained in culture and passaged accordingly. Immunofluorescent staining was performed to identify nuclei DAPI (blue), H2A.X (s139) (green), and F-actin (Rhodamine-phalloidin-red) in HEMn-LP and HEMn-DP cells at 1 day after single exposure and at 28 days after intermittent exposure. (Bottom left) Cyclobutane pyrimidine dimer (CPD) formation was quantified by ELISA (Cell BioLab). At 1 day after single UVB exposure, CPD formation was similar in both HEMn-LP and HEMn-DP cells. At 28 days after intermittent exposure, the relative change in CPD formation was significantly decreased in HEMn-LP cells compared with HEMn-DP cells (*P < 0.05). (Bottom right) Quantitation of the H2A.X signal was performed with the ImageJ software (NIH). At 1 day after single UVB exposure H2A.X foci formation was similar in both HEMn-LP and HEMn-DP cells. At 28 days after intermittent exposure, HEMn-LP retained significantly higher levels of H2A.X foci compared with HEMn-DP (*P < 0.05) (scale bar = 10 μm). (b) HEMn-LP and HEMn-DP cells were either sham-exposed (top row) or exposed to UVB (middle row). UVB exposure was either single dose UVB (100 mJ/cm2) (1 day) or intermittent UVB (100 mJ/cm2 at 14 days) or intermittent UVB and maintained in culture for an additional 14 days (28 days). Cells were fixed and stained for expression of senescence-associated beta galactosidase (SA-β-gal). (Upper two panels) The corresponding cell morphology of UVB-exposed cells at each time point is shown by phase-contrast microscopy in the lower panel. Quantification based on SA-β-gal staining was performed by cell counts in triplicate and calculation of the percentage of positively stained cells (scale bar = 50 μm). (c) HEMn-LP and HEMn-DP cells were treated as in (b), cell lysates were prepared, and western blot was performed for protein expression of p21, p53, p16, STAT1 and pSTAT, MX1, AKT and pAKT, p-p38, and pErk 1/2. β-Tubulin was used as control. (d) After intermittent exposure, HEMn-LP and HEMn-DP cells were maintained for another 14 days in culture (28 days). Cells were then collected and mRNA was extracted. qPCR was performed for gene expression of genes that were differentially regulated at 14 days. (e) Intermittent UVB-treated HEMn-LP and HEMn-DP cells were treated with doxorubicin (1–10 μg/ml) for 2 days after last exposure. Cell viability was determined by the MTT assay. (f) Intermittent UVB-treated HEMn-LP cells were treated with high doses of UVB (300 and 600 mJ/cm2) 48 hours after the last UVB exposure. Twenty-four hours after high-dose exposure, cells were collected and stained with the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & PI. The apoptotic and dead cells were detected by FACS analysis (upper panel). The quantification data are shown in graphic form based on FACS data of three independent experiments. AKT, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; ERK, extracellular signal-regulated kinase; STAT1, signal transducer and activator of transcription 1.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

5. Figure 3
Senescence associated secretory phenotype acts in a paracrine manner to induce IFN stimulatory gene response in UVB-naïve HEMn-LP. (a) Cell culture supernatants from intermittent UVB-treated HEMn-LP and HEMn-DP were collected 24 hours after the last UVB treatment. Naïve HEMn-LP cells plated in six-well plates were then treated with cultured supernatants. Twenty-four hours after treatment, cells were collected and mRNA was extracted. qPCR determination for gene expression of IL6, MX1, OAS2, IRF7, and STAT1 was performed. (b) IL6 secretion (pg/ml) was determined by ELISA from cell culture supernatants of either single dose (24 hours) or intermittent UVB-treated HEMn-LP cells at indicated times. (c) HEMn-LP (upper panel) and HEMn-DP (lower panel) cells were treated with IL6 (20 ng/ml) for 24 or 48 hours. Cells were collected, mRNA was extracted, and qPCR was performed for gene expression of IL6, MX1, OAS2, IRF7, STAT1, and STAT2. STAT, signal transducer and activator of transcription.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

6. Figure 4
UVB induces HMGB1 expression and JAK1/2 activation in HEMn-LP cells. (a) HEMn-LP cells were exposed to a single dose of UVB (100 mJ/cm2). At varying time points after exposure (0–72 hours), cells were collected and protein was extracted. Western blot was performed for NFkB, HMGB1, HMGB2, S100A, and RAGE protein expression. (b) HEMn-LP cells were exposed to a single dose of UVB (100 mJ/cm2) (left panel) or intermittent UV (right panel). Sham-irradiated cells served as control. At varying time points after single dose or intermittent exposure, cell culture supernatants were collected (0–72 and 24 hours, respectively). Extracellular HMGB1 protein levels (ng/ml) were determined by ELISA in both single exposure cells (left panel) and intermittent exposure cells (right panel). (c) Twenty-four hours after the last UVB exposure, intermittent UVB-treated HEMn-LP cells were treated with or without the JAK inhibitor ruxolitinib (300 nM) for 24, 48, and 72 hours. At varying time points after treatment, cells were collected, protein was extracted, and western blot was performed to determine protein expression of MX1, NFkB, JAK1 and 2, STAT, HMGB1 MX1, and RAGE. Actin protein was served as control. (d) Twenty-four hours after the last UVB exposure, intermittent UVB-treated HEMn-LP cells were treated with or without the JAK inhibitor ruxolitinib (300 nM) for 24 hours. Cells were collected, mRNA was extracted, and quantitative real-time reverse transcriptase-PCR was performed for MX1, OAS2, IRF7, and STAT1. ERK, extracellular signal-regulated kinase; HMGB1, high-mobility group box 1; RAGE, receptor for advanced glycosylation end product; STAT, signal transducer and activator of transcription.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

7. Figure 5
HMGB1/RAGE engagement contributes to UVB-induced SASP. (a) HEMn-LP cells were infected with lentiviruses expressing using two different shRNA targeting RAGE (shRAGE #1, #2) or control scrambled shRNA (SCR). Infected cells were then either sham-exposed or intermittently exposed to UVB. Twenty-four hours after the last UVB exposure, cells were collected and protein was extracted. Western blot was performed for protein expression of RAGE, STAT1, MX1, and HMGB1. β-Tubulin served as control. (b) HEMn-LP cells were infected with lentiviruses expressing shRNA targeting RAGE (shRAGE) or control scrambled shRNA (SCR). Infected cells were treated with or without recombinant active HMGB1 protein for 24 hours. After treatment, cells were collected and protein was extracted. Western blot was performed for protein expression of RAGE, JAK1 and 2, p-JAK1 and 2, Erk1/2, pERK1/2, and MX1. β-Tubulin served as control. (c) HEMn-LP cells were infected with lentiviruses expressing shRNA targeting RAGE or control scrambled shRNA (SCR). Infected cells were treated with intermittent UVB. Twenty-four hours after the last UVB exposure, cells were collected, mRNA was extracted, and quantitative real-time reverse transcriptase-PCR was performed for gene expression of RAGE, MX1, and IRF7. (d) HEMn-LP cells were infected with lentivirus shRNA targeting RAGE (shRAGE) or control scrambled shRNA (shSCR). Infected cells were treated with intermittent UVB. Forty-eight hours after the last UVB exposure, cells were treated with high doses of UVB (300 and 600 mJ/cm2). Twenty-four hours after high-dose exposure, cells were collected and stained with Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & PI. The apoptotic and dead cells were detected by FACS analysis. The quantification data are shown in graphic form based on FACS data of three independent experiments. (e) Hypothetical model representing the regulation of intermittent UVB-induced DNA damage response in melanocytes. Upregulation/activation of damage associated molecular patterns such as HMGB1, S100A, and their receptor RAGE result in the activation of the JAK/STAT1 pathway that leads to an increase in para-inflammatory response and cell resistant to UVB-induced apoptosis. Loss of receptor RAGE blocked the activation of JAK/STAT1 signaling. JAK inhibitors block the upregulation of RAGE in a feedback regulation. DDR, DNA damage response; ERK, extracellular signal-regulated kinase; HMGB1, high-mobility group box 1; ISG, IFN stimulatory gene; RAGE, receptor for advanced glycosylation end product; SASP, senescence associated secretory phenotype; SIR, senescence-associated inflammatory response; STAT, signal transducer and activator of transcription.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

8. Figure 5
HMGB1/RAGE engagement contributes to UVB-induced SASP. (a) HEMn-LP cells were infected with lentiviruses expressing using two different shRNA targeting RAGE (shRAGE #1, #2) or control scrambled shRNA (SCR). Infected cells were then either sham-exposed or intermittently exposed to UVB. Twenty-four hours after the last UVB exposure, cells were collected and protein was extracted. Western blot was performed for protein expression of RAGE, STAT1, MX1, and HMGB1. β-Tubulin served as control. (b) HEMn-LP cells were infected with lentiviruses expressing shRNA targeting RAGE (shRAGE) or control scrambled shRNA (SCR). Infected cells were treated with or without recombinant active HMGB1 protein for 24 hours. After treatment, cells were collected and protein was extracted. Western blot was performed for protein expression of RAGE, JAK1 and 2, p-JAK1 and 2, Erk1/2, pERK1/2, and MX1. β-Tubulin served as control. (c) HEMn-LP cells were infected with lentiviruses expressing shRNA targeting RAGE or control scrambled shRNA (SCR). Infected cells were treated with intermittent UVB. Twenty-four hours after the last UVB exposure, cells were collected, mRNA was extracted, and quantitative real-time reverse transcriptase-PCR was performed for gene expression of RAGE, MX1, and IRF7. (d) HEMn-LP cells were infected with lentivirus shRNA targeting RAGE (shRAGE) or control scrambled shRNA (shSCR). Infected cells were treated with intermittent UVB. Forty-eight hours after the last UVB exposure, cells were treated with high doses of UVB (300 and 600 mJ/cm2). Twenty-four hours after high-dose exposure, cells were collected and stained with Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & PI. The apoptotic and dead cells were detected by FACS analysis. The quantification data are shown in graphic form based on FACS data of three independent experiments. (e) Hypothetical model representing the regulation of intermittent UVB-induced DNA damage response in melanocytes. Upregulation/activation of damage associated molecular patterns such as HMGB1, S100A, and their receptor RAGE result in the activation of the JAK/STAT1 pathway that leads to an increase in para-inflammatory response and cell resistant to UVB-induced apoptosis. Loss of receptor RAGE blocked the activation of JAK/STAT1 signaling. JAK inhibitors block the upregulation of RAGE in a feedback regulation. DDR, DNA damage response; ERK, extracellular signal-regulated kinase; HMGB1, high-mobility group box 1; ISG, IFN stimulatory gene; RAGE, receptor for advanced glycosylation end product; SASP, senescence associated secretory phenotype; SIR, senescence-associated inflammatory response; STAT, signal transducer and activator of transcription.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions