Duolink 2 fluorescence

The PLA was performed as described in the manufacturer’s protocol (Duolink II Fluorescence, OLink, Upsalla, Sweden). Briefly, HUVECs were fixed in phosphate buffered formaldehyde solution (4%), permeabilized with Triton X-100 (0.2%), blocked with serum albumin solution (3%) in phosphate-buffered saline, and incubated overnight with Anti-dsDNA [35I9 DNA] (Abcam, ab27156, 1:500), Anti-MPP8 (Bethyl, A303-051A-M, 1:500), Anti-H3K9me3 (Diagenode, SN-146-100, 1:500) or Anti-SETDB1 (Santa Cruz Biotechnology, ESET (G-4): sc-271488, 1:500). Samples were washed and incubated with the respective PLA-probes for 1 h at 37 °C. After washing, samples were ligated for 30 min (37 °C). After an additional washing step, the amplification with polymerase was performed for 100 min (37 °C). The nuclei were stained using DAPI. Images (with Alexa Fluor, 546 nm) were acquired by confocal microscopy (LSM 510, Zeiss) using the ZEN 3.2 software.

Proximity ligation analysis (PLA) was performed according to the manufacturer's protocol (Duolink II Fluorescence, Duolink In Situ Detection Reagents Orange, OLink). Cells were fixed in 4% phosphate-buffered formaldehyde solution, permeabilized with 0.05% Triton X-100, blocked, and incubated overnight with rabbit monoclonal antibodies against NOX4 (provided by one of the co-authors (P.J.-D.)) and CANX antibody (Santa Cruz, number sc-6465) as well as negative and positive controls to validate the assay (data not shown). To show the specificity of the CANX antibody, a blocking peptide was used (Santa Cruz, number sc-6465 P). Samples were washed, incubated with the respective PLA probes for 1 h (37 °C), washed, and ligated for 30 min (37 °C). After an additional washing, amplification with polymerase was performed for 100 min (37 °C). The nuclei were stained with DAPI. Images were acquired by confocal microscope (LSM 510, Zeiss) and Plan-Neofluar ×40/1.3 oil objective at room temperature. Acquisition software Zen 2009 (Zeiss) was used. For each biological sample (n ≥ 3) eight pictures were evaluated and counted. Quantitative analysis was performed using Fiji software. Interactions were normalized to nuclei.

PLA (Duolink II Fluorescence, Olink Bioscience) was used for the detection of protein-protein interactions according to the manufacturer's instructions. Briefly, adherent cells grown on round glass slides in 24-well plates were fixed for 10 min with 4% paraformaldehyde, permeabilized in PBS containing 0.5% Triton X-100 for 20 min at room temperature, and then blocked by incubation in PBS containing 7.5% goat serum and 7.5% foetal bovine serum for 1 h at room temperature. Next, the cells were incubated overnight at 4°C with two antibodies against the two proteins of interest. The following primary antibodies were used: TRF2 (1:100, IMG-124, Imgenex), PCAF (1:100, AB9962, Millipore), GCN5 (1:100, H-75, Santa Cruz), TRF1 (1:100, ab10579, Abcam), and P300 (1:100, NA 46, Calbiochem). The slides were then washed three times with 0.1% Triton X-100 in PBS and incubated with two PLA probes (PLA probe MINUS stock and PLA probe PLUS stock; Duolink II) for 1 h at room temperature. Subsequently, the slides were washed three times with 0.1% Triton X-100 in PBS. Ligation was conducted for 30 min at 37°C, followed by two washes with 0.1% PBS and Triton X-100 and amplification using DNA polymerase (Duolink II) for 100 min at 37°C. The slides were then washed, dried and mounted with Dapi-Fluoromount-G (Southern Biotech). Images were captured using a Leica DM 2500 microscope.