Journal: Scientific reports

Article Title: IL-1α is a DNA damage sensor linking genotoxic stress signaling to sterile inflammation and innate immunity.

doi: 10.1038/srep14756

Figure Lengend Snippet: Figure 1. IL-1α is recruited to DNA damage sites and secreted after genotoxic stress. (a) Human HT1080 fibrosarcoma and HaCaT human keratinocytes were subjected to genotoxic stresses: UV irradiation (5 mJ/cm2), 10 mM H2O2 or 50 μ gr/ml Bleomycin. 16 h post exposure, hIL-1α ELISA was used to measure secreted IL-1α in cell supernatants. All experiments were performed in triplicates and data are expressed as mean ± SD. (b) Nuclear/cytoplasmic re-localization of IL-1α after DNA damage. Live cell imaging of B16 melanoma cells expressing GFP-IL-1α during treatment with 100 μM H2O2. Images were collected every 30 min for a period of 24 h. Representative images from indicated time points are shown (for full video see Supplementary Video 1, for averaged fluorescence intensities see also Supplementary Figure 1b) White scale bars, 20 μm. (c,d) Nuclear IL-1α co-localizes with γ H2AX foci after genotoxic stress. (c) B16 melanoma cells expressing GFP-IL-1α were treated with Etoposide 10 µg/mL for 2 h or (d) microirradiated with femtosecond laser pulses at λ = 775 nm (see also Supplementary Videos 2 and 3). After fixation of cells, detection of GFP-IL-1α , DAPI or immunostaining of γ H2AX was preformed and visualized by confocal microscopy. White scale bars, 20 μm (e) Recruitment kinetics of IL-1α to DNA damage sites. B16 melanoma cells expressing IL-1α –GFP were laser-microirradiated along a single line to induce DNA damage. Fluorescence intensity in the damaged region was measured up to 15 min from irradiaton in 1 min intervals. Data is expressed as mean ± SEM of increase in fluorescence intensity (n = 10 cells). (f) IL-1α localizes to Cyclobutane Pyrimidine Dimers (CPD) induced via laser microirradiation. B16 melanoma cells expressing GFP-IL-1α were laser irradiated and CPDs were visualized by immunostaining using specific antibodies. White scale bars, 20 μm.

Article Snippet: The cells were allowed to express GFP-IL-1α and left to recover for an additional 24 h. To induce DNA damage, UV was applied as described above and levels of secreted GFP-IL-1α were measured by measuring GFP using the PathScan total GFP Sandwich ELISA kit (Cell signaling).

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Expressing, Fluorescence, Immunostaining, Confocal Microscopy

Journal: Scientific reports

Article Title: IL-1α is a DNA damage sensor linking genotoxic stress signaling to sterile inflammation and innate immunity.

doi: 10.1038/srep14756

Figure Lengend Snippet: Figure 3. IL-1α acetylation within the nuclear localization sequence impacts on IL1α subcellular localisation. (a) IL-1α precursor is recognized by a pan acetyl antibody. Endogenous IL-1α was immunoprecipitated (IP) from nuclear extracts of Raw 264.7 cells, either induced or non-induced with 100 ng/ml LPS. Total IP proteins were separated over 15% SDS PAGE, transferred to nitrocellulose membranes and blotted with anti-mouse IL-1α (top panel) or anti-Kac (bottom panel). Acetylated IL- 1α is marked by arrows and IP antibody light and heavy chain signals are indicated. (b) Annotated MS/ MS spectrum of the tryptic peptide VTVSATSSN(Deam)GK(Acetyl)ILK (MH2 + 724.40 Da) showing acetylation of IL-1α (Uniprot ID P01582) at K82 and N80 deamidation. (c) PrecIL-1α K82 mutants affect IL-1α sub-cellular localization. Confocal microscopic analysis of GFP tagged WT IL-1α and mutations of precIL-1α K82 to glutamine (precIL-1α K82Q, mimicking acetylation) and to arginine (precIL-1α K82R non-acetylateable). White scale bars, 20 μm (d) IL-1α K82 mutations reduce cytokine secretion after DNA damage. Mouse B16 cells were transfected with the indicated GFP IL-1α plasmids. The cells were then subjected to 100 μ M H2O2. 16h after stress induction levels of secreted GFP IL-1α in cell growth medium was measured using a GFP ELISA. GFP IL-1α levels in cell lysates were used to normalize for transfection efficiencies and non-transfected cells were used as negative controls. Data are expressed as mean ± SD of three independent experiments. (e) Histone deacetylase inhibition by TSA increases IL-1α nuclear localization. Images of cells expressing GFP IL-1α either non-treated (control) or treated with TSA (100 ng/ml) were collected every hour for 22 h and representative images for three time points (0, 11 and 22 hours) are shown (For averaged fluorescence intensities of nuclear/cytoplasmic ratios see Supplementary Figure 1b). (f) HDAC-1 and IL-1α can co-localize at DNA damage lesions. Cells expressing GFP IL-1α were laser- microirradiated for the induction of DNA damage. Localization of HDAC-1 and IL-1α –GFP were visualized by confocal microscopic analysis.

Article Snippet: The cells were allowed to express GFP-IL-1α and left to recover for an additional 24 h. To induce DNA damage, UV was applied as described above and levels of secreted GFP-IL-1α were measured by measuring GFP using the PathScan total GFP Sandwich ELISA kit (Cell signaling).

Techniques: Sequencing, Immunoprecipitation, SDS Page, Tandem Mass Spectroscopy, Transfection, Enzyme-linked Immunosorbent Assay, Histone Deacetylase Assay, Inhibition, Expressing, Control, Fluorescence