Anti trimethyl histone h3 lys9

Cells were pre-extracted with 0.05% or 0.25% Triton X-100 in PBS for 5 min on ice, fixed in 4% paraformaldehyde in PBS for 10 min at RT, washed with PBS, post-extracted in 0.25% Triton X-100 in PBS for 10 min at RT and blocked with 3% bovine serum albumin (BSA) in PBS. The cells were subsequently incubated at 37°C for 1 h with anti-γH2AX (Cell Signaling Technology, #9718), anti-RPA2 (Abcam, ab2175), anti-Mre11 (GeneTex, GTX70212), anti-trimethyl-histone H3 (Lys9) (Merck, 07442) or anti-HP1β (Cell Signaling Technology, #8676) diluted in PBS containing 3% BSA and 0.05% Tween 20. The cells were subsequently washed three times and incubated with secondary antibodies conjugated to the appropriate fluorophore (Alexa-488 or Alexa-594) for 1 h at RT. After three additional washes, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted with Vectashield (Vector Laboratories). Proximity ligase assays (PLAs) were performed with the indicated primary antibodies diluted 1:500 (SMARCAL1 and HP1α; Merck, 05-689) and Duolink In Situ PLA kits (Sigma-Aldrich). Immunofluorescence images were obtained with a fluorescence microscope (BZ-9000, Keyence) or a confocal laser scanning microscope (TCS SP8, Leica).

We detected senescence-associated heterochromatin foci (SAHFs) as described previously with minor modifications (47 (link)). Cells prepared in chamber slides were fixed in 4% paraformaldehyde for 10 min and then permeabilized with 0.2% Triton-X 100 for 5 min. After blocking cells in 3% BSA for 5 min, the cells were immunostained with anti-trimethyl-histone H3 (Lys9) (1:500; Merck Millipore, 07-422) as the primary antibody for 2 h, followed by Alexa Fluor 594-conjugated anti-rabbit IgG (1:1000; Invitrogen) as the secondary antibody for 1 h. To stain DNA, cells were incubated in 0.15 μg/ml DAPI for 3 min. After washing, cells were soaked in fluorescence mounting medium (Dako) and covered with a coverslip. SAHFs were observed on a BZ-X810 All-in-One Fluorescence Microscope (Keyence).