Anti gamma h2ax

Antibodies employed for Western blot analysis or immunofluorescence labeling were: anti-lamin A/C (goat polyclonal, Santa Cruz SC-6215) and anti-prelamin A (goat polyclonal, Santa Cruz SC-6214); anti-farnesyl-prelamin A, (rabbit polyclonal, Diatheva 1188-2), raised against the last 15 aminoacids of the prelamin A sequence including the farnesylated cysteine residue but not the SIM sequence [65 (link)]; anti-prelamin A, (rabbit polyclonal, Diatheva 1188-1), raised against the last 18 aminoacids of the prelamin A sequence [65 (link)]; anti-LAP2alpha (rabbit polyclonal, [66 (link)]); anti-trimethyl-H3K9, rabbit polyclonal and anti-acetyl-H3K9, rabbit polyclonal (Upstate); anti-emerin, mouse monoclonal (Monosan); anti-LC3-B, rabbit polyclonal (Cell Signaling Technologies); anti- 53BP1 antibody (Cell Signaling); anti-gammaH2AX (Abcam); anti-Oct-1 (Santa Cruz); anti-actin, goat polyclonal (Santa Cruz), anti-phospho-Lamin A Ser404 rabbit polyclonal antibody [26 (link)].

Cells were first washed with Phosphate Buffer Saline (Thermo Fisher) and fixed with 4% Paraformaldehyde (MS). Cells were stained with the following primary antibodies and concentrations, MYHC‐IIb eFluor 660 (50‐6503‐32; Thermo Fisher; 1:100), α‐actinin (sc‐7453; Santa Cruz; 1:500), and myogenin (sc‐576; Santa Cruz; 1:200). Anti‐PARP‐1 (Santa, SC‐8007), anti‐XRCC1 (Cell Signalling, 2735S), anti‐gamma H2AX (Abcam, 2893), anti‐53BP1 (Abcam, ab175933), and anti‐Osteocalcin (Santa, SC‐365797).The following secondary antibodies were also used together with non‐conjugated primary antibodies, Goat‐anti‐mouse Alexa Fluor 488 (A11001; Thermo Fisher; 1:500), Goat‐anti‐rabbit Alexa Fluor 594 (A11012; Thermo Fisher; 1:500), and Goat‐anti‐mouse Alexa Fluor 647 (A21235; Thermo Fisher; 1:500). 4'6‐Diamidino‐2‐Phenylindole (d9542; Sigma) was used as a nuclear counter stain according to manufacturer's recommendations. Stained cells were imaged with a Zeiss fluorescence microscope.

Total cell lysates containing equal amounts of proteins were loaded onto 4–12% gels for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and then transferred to Immobilon-P Transfer Membrane (Millipore, Bedford, MA). The membranes were blocked with 5% non-fat skim milk dissolved in PBS containing 0.02% Tween-20 and incubated overnight at 4 °C with specific primary antibodies. The membranes were subsequently incubated with specific horseradish peroxidase conjugated secondary antibodies. Protein bands were visualized using a Fusion FX5 system (Vilber Lourmat, Eberhardzell, Germany). The following primary antibodies were used: anti-Rad51 (Santa Cruz Biotechnology, CA, USA), anti-DNMT1 (Santa Cruz Biotechnology), anti-DNMT3a (Abcam, Cambridge, MA, USA), anti-DNMT3b (Sigma-Aldrich, St. Louis, MO, USA), anti-gamma H2AX (Abcam), and anti-GAPDH (Advanced ImmunoChemical Inc., Long Beach, CA).

A549 cells were cultured on glass coverslips under the cell culture treatment conditions described above for the MTT assay. On day 7, coverslips were transferred to a solution of 4% paraformaldehyde in PBS and fixed for 15 min at room temperature. Cells were then permeabilized with 0.2% Triton X-100, 0.125 M glycine in PBS for 12 min at room temperature. Blocking followed for 1 h at room temperature with 2% BSA, 2% horse serum and 0.1% Triton X-100 in PBS. γ-H2AX foci were detected using anti-gamma H2A.X (Abcam, ab11174) diluted 1:1000 in blocking buffer and incubated at 4 °C for 16 h. Cells were washed three times with PBS for 10 min and incubated with secondary antibody (AlexaFluor anti-mouse 488 nm) diluted 1:400 for 45 min in the dark at room temperature. Cells were then washed three times for 10 min with PBS. Finally, coverslips were inverted and mounted onto a microscope slide with Immu-Mount (Fisher Scientific) and DAPI for counterstain. Slides were imaged on a Zeiss Axio Imager M1 microscope (Carl Zeiss, Thornwood NY), and resulting images were analyzed using Zeiss' ZEN Digital imaging suite software. A minimum of 200 cells per treatment condition were imaged, and cells with > 5 γH2AX foci were quantified and divided by total number of cells as determined by DAPI counterstain.

Cell cycle analysis was done using a Gallios flow cytometer (Beckman Coulter) and PI. After treatment, cells were washed twice in ice-cold PBS, trypsinized and fixed in 90% Methanol overnight at -20°C, permeabilized with PBS containing 0.25% Triton X-100 for 20 min at 4°C, blocked with 10% BSA in PBS and incubated with 1 μg of anti-phospho-histone H3 (Millipore, #16–218) or anti-gammaH2AX (Abcam, ab22551) per 10⁶ cells for 60 min on ice. Following washing, cells were incubated with FITC-conjugated secondary antibody (Millipore, #12–506) for 30 min on ice, washed, and resuspended in PBS containing RNase and 50 μg/ml PI prior to analysis.