Axiovert 200 imaging system

HEK (human embryonic kidney)-293T cells were purchased from the A.T.C.C. (Manassas, VA, U.S.A.) and maintained in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (Gibco) supplemented with 10 % FBS. Cells (5 × 10⁵) were seeded in each well of a six-well plate in 2 ml of medium. Inhibitors were added to each well at a final concentration of 0.2, 1, 5, 10 or 15 μM in the presence of 1 % DMSO. For the control sample only 1 % DMSO was added to the well. Cells were trypsinized after 48 h and deposited on to microscope slides by cytospin (200 rev./min for 10 min). For GFP visualization, cells were transfected using Lipofectamine^™ 2000 according to the manufacturer’s protocol (Invitrogen). Transfected HEK-293T cells were FACS-sorted on the basis of GFP intensity (BD FACSAria II). Cytospinning, fixation and immunofluorescence were performed as described previously [27 (link)]. The primary antibodies used for immunofluorescence include anti-H3K9me3 (catalogue number 07-523; Millipore), anti-H4K20me3 (histone H4 trimethylated at Lys²⁰; catalogue number 39180; Active Motif) and anti-HP1γ (catalogue number MAB3450; Millipore). Images were collected on a Zeiss Axiovert 200 imaging system equipped with an Axiocam MR digital camera controlled by AxioVision software or on a Zeiss LSM Pascal confocal microscope.