Journal: Journal of Biomedical Science

Article Title: Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex

doi: 10.1186/s12929-023-00931-5

Figure Lengend Snippet: ET-1 induced a decrease in HDAC2 activity and acetylation of H3. A Cells were stimulated with ET-1 for 5, 10, or 30 min. The cell lysates were immunoprecipitated with antibody specific for HDAC2, and then HDAC activity was detected. Bars indicate values of the mean ± SEM ( n = 3). * p < 0.05 versus control group. B Cells were stimulated with ET-1 for 5, 10, 20, 30, or 60 min, and then the acetylation of histone H3 was evaluated using Western blotting. H3 was used as a loading control. Bars indicate mean ± SEM ( n = 4). * p < 0.05 versus control group. C Cells were transfected with either 0.5 or 1 μg of HDAC2-HA plasmid for 24 h and then treated with ET-1 for 20 min. Cells were subsequently lysed and immunoblotted with antibodies specific for histone H3, acetyl-H3, or HA. Bars indicate values of the mean ± SEM ( n = 4). * p < 0.05 versus ET-1-treated group. D Cells were transfected with 1 μg of HDAC7-GFP plasmid for 24 h and then treated with ET-1 for 20 min. Cells were subsequently lysed and immunoblotted with antibodies specific for histone H3, acetyl-H3, or GFP. Bars indicate values of the mean ± SEM ( n = 5)

Article Snippet: Antibodies specific for Sin3A, MeCP2, and GFP-tag, were purchased from Cell Signaling Technology (Danvers, MA, USA), and the anti-α-tubulin antibody was purchased from Transduction Laboratories (Lexington, KY, USA).

Techniques: Activity Assay, Immunoprecipitation, Control, Western Blot, Transfection, Plasmid Preparation

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: ( a ) Human HT1080 fibrosarcoma and HaCaT human keratinocytes were subjected to genotoxic stresses: UV irradiation (5 mJ/cm 2 ), 10 mM H 2 O 2 or 50 μgr/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 μ M H 2 O 2 . Images were collected every 30 min for a period of 24 h. Representative images from indicated time points are shown (for full video see , for averaged fluorescence intensities see also ) White scale bars, 20 μ m. ( c , d ) Nuclear IL-1α co-localizes with γH2AX 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 and ). After fixation of cells, detection of GFP-IL-1α, DAPI or immunostaining of γH2AX was preformed and visualized by confocal microscopy. White scale bars, 20 μ m ( 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 μ m.

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: ( 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 μ m ( 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 μM H 2 O 2. 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 ). ( 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: Immunoprecipitation, SDS Page, Tandem Mass Spectroscopy, Transfection, Enzyme-linked Immunosorbent Assay, Histone Deacetylase Assay, Inhibition, Expressing, Control, Fluorescence