Journal: Cell Death & Disease
Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns
Figure Lengend Snippet: Release of nucleosomes and DAMPs from amino-acid-depleted HeLa cells. ( A ) An inverted microscopic image of HeLa cells in the condition of amino-acid depletion. Arrows designate dying HeLa cells. ( B ) Genomic sequences of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), Fas, cytochrome oxidase subunit 1 ( Co1 ) and ATP synthase subunit 6 ( ATP6 ) were PCR amplified from extracellularly released DNA, genomic or mitochondrial DNAs. ( C ) Inverted and fluorescent microscopic images were taken from amino-acid-deprived HeLa cells in the presence of SYTOX, a membrane-impermeable DNA dye. HeLa cells deprived of amino acids for 15 h were fluorescence stained with histone H1 or IL6 antibodies ( D ), histone H2A, H2B, H3 or H4 antibodies ( E ) or HMGB1, Hsp90 or ERp57 antibodies ( F ) in combination with histone H1 antibody and 4',6-diamidino-2-phenylindole (DAPI). ( G ) Amino-acid-deprived HeLa cells were stained with SYTOX to determine viability, fixed and stained with DAPI and histone H1 antibodies. ( H ) Amino-acid-deprived HeLa cells were untreated or treated with MNase (500 mU/ml) for 10 min. Released DNA was quantitated at the indicated times. Data from triplicate samples are presented as mean±S.D. ( I ) Conditioned media from amino-acid-deprived HeLa cells treated or untreated with MNase were western blotted with histone H1, 2B, H3, H4, IL6, ERp57, HMGB1 or Hsp90 antibodies. ( J ) Images captured every hour from live imaging of amino-acid-deprived HeLa cells with SYTOX (green) and DRAQ5, membrane-permeable DNA dye (red). ( K ) SYTOX fluorescent intensities were measured from circularized areas of live imaging of amino-acid-deprived cells in 5-min intervals. ( L ) TEM images of control cells ( L a) and amino-acid-deprived HeLa cells ( L b– L d). ( M ) Amino-acid-deprived HeLa cells were fluorescence stained with lamin and nuclear pore antibodies, or lamin antibody and wheat germ agglutinin (WGE)
Article Snippet: Released DNA from dying cells were digested with 500 mU/ml MNase (Thermo Scientific) for 5 min. Nuclease activity was stopped with 5 mM EDTA and the culture supernatants were collected and stored at −20 °C until quantification.
Techniques: Genomic Sequencing, Polymerase Chain Reaction, Amplification, Fluorescence, Staining, Western Blot, Imaging, Transmission Electron Microscopy