Phospho γh2a x ser139

Phosphorylated p53 and histone H2A.X (γH2A.X) in nuclei and CoxIV in mitochondria were detected by immunofluorescence. Cells grown in 48‐well plates were fixed with 4% w/v formaldehyde in PBS for 10 minute before blocking and permeabilization with 0.15% v/v Triton X‐100, 10% v/v goat serum and 1% w/v BSA in PBS for 10 minute. Cells were then incubated with anti‐phospho‐p53 (Ser15) (#9284, Cell Signaling Technology) or ‐phospho‐γH2A.X (Ser139) (#9718, Cell Signaling Technology) rabbit polyclonal IgG and anti‐CoxIV (#11967 Cell Signaling Technology) mouse monoclonal IgG, overnight at 4°C. After washing, cells were incubated with Alexa Fluor 555 anti‐rabbit‐ and 488 anti‐mouse conjugates (#4413 and #4408, respectively, Cell Signaling Technology) for 1 hour at room temperature. All antibodies were used at a 1 in 200 dilution. Cells were counterstained with DAPI (1 μg/mL, Sigma) and mounted in 70% v/v glycerol. Fluorescent images of cells were taken at 100× magnification using a Nikon Eclipse Ti‐U fluorescence microscope.

Cells grown on glass coverslips were rinsed in PBS, fixed in 3.7% formaldehyde in PBS, permeabilized in 0.5% Triton X-100, and then blocked with 20% normal goat serum in PBS for 1 h at RT. Coverslips were processed for immunofluorescence using the following antibodies: Phospho-γH2AX (Ser139, Rabbit, #4353, 1:200), Tri-Methyl-Histone H3 (Lys9, Rabbit #1396, 1:400), both from Cell Signaling, Danvers MA, USA. Secondary antibodies conjugated to Alexa Fluor 488 and Cy3 were also used. DNA was counterstained with 0.05 μg/ml DAPI.
Detection of autophagic structures was obtained by fluorescence microscopy observing LC3B puncta in H1299-EGFP-LC3B expressing cells. Typically, at least 100 cells were counted, and cells with more than 10 puncta were considered autophagy positive. Preparations were examined under either an Olympus AX70 microscope, using a 40x or 100×/1.35 NA objective, or a Nikon Eclipse 90i microscope. Single-cell images were taken using an immersion oil 100x or 40x objective. Images were processed using the maximum intensity projection method and the “spot detection” and “count objects” tools of NIS-Element AR 5.02 were used. Automated quantification of H3K9me3 fluorescence intensity signals was employed: images were analyzed using open-source Cell Profiler 4.1.3 image analysis software (https://cellprofiler.org/) to measure fluorescence-integrated intensity values.