Falcon chambered cell culture slides

Cells were cultured in black 96-well plates (Corning) or Falcon™ chambered cell culture slides (four- or eight-well, Nunc, Penfield), fixed in 10% formalin (Sigma) for 10 min at room temperature (RT), washed with 1× PBS and permeabilized in 1× PBS–0.25% Triton™ X-100 (Sigma) for 10 min. Wells or slides were washed with 1× PBS and unspecific antibody binding was blocked for 1 h using 1% bovine serum albumin (BSA, IgG/protease-free; Jackson ImmunoResearch) and 4% normal donkey serum (Sigma) diluted in 1× PBS (blocking solution). Primary antibodies (Supplementary Table S2) were diluted in blocking solution and incubated with fixed cells overnight at 4°C. Cells were washed three times in 1× PBS–0.05% Tween™ 20 (Fisher Scientific) and incubated with secondary antibodies for 1 h at RT. Cells were washed three times in 1× PBS–0.05% Tween™ 20 and slides were mounted with ProLong™ Gold Antifade Reagent with DAPI (Life Technologies), while nuclei of cells in 96-well plates were stained using DAPI solution (0.5 μg/ml) in 1× PBS. Images were acquired with a Carl Zeiss AxioObserver Z1 fluorescence microscope (Jena, Germany) using a 20× objective and quantifications were performed with AxioVision and ImageJ softwares. The number of foci per nucleus was analyzed in >200 nuclei for each condition.

Cells were cultured in four-well Falcon™ chambered cell culture slides (Nunc, Penfield) and pulsed with 10 μM of BrdU for 24 or 72 h, and then fixed in 10% formalin (Sigma) for 10 min at RT and permeabilized in 1× PBS–0.25% Triton™ X-100 (Sigma) for 10 min. Genomic double-stranded DNA was then enzymatically denatured using 10 U/ml DNase I (Roche) and 400 U/ml EXO III (New England Biolabs) for 30 min at 37°C. To stain BrdU, the previously described immunofluorescence protocol was used.

HASMCs cultured in Falcon Chambered Cell Culture Slides (Fisher, 08774208) were fixed in 4% paraformaldehyde in PBS for 15 minutes at room temperature. After washing with PBS 3 times, slides were permeabilized in a permeabilizing solution (0.1% Triton X-100 and 0.5% BSA in PBS) for 10 minutes at room temperature and incubated in blocking buffer (5% donkey serum in PBS) for 1 hour at room temperature. Next, slides were stained with primary Abs against P65 (1:100, CST, catalog 8242) in dilution buffer (5% normal donkey serum in PBS) overnight at 4°C. After washing with PBS 3 times, slides were incubated with Alexa Fluor–conjugated secondary Abs (Jackson ImmunoResearch Laboratories) and subsequently mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen, P36935) before images were captured with an Olympus IX73 microscope.