Duolink in situ pla reagents

Duolink^®in situ PLA reagents (Sigma-Aldrich Inc.) were used to detect the interaction between β-catenin and BCL9 and the manufacturer’s protocol was followed. Briefly, C2C12 cells, cultured on chamber slides to 60–70% confluence, were treated with Wnt3a (100ng/mL for 4 hours) ± SetD7 inhibitor PFI-2 (5µM for the last 2 hours). Cells were then fixed, permeabilized and incubated overnight with mouse anti-β-catenin (Santa Cruz) and rabbit anti-BCL9 (Thermo Fisher). The following day, cells were incubated with secondary antibodies conjugated to oligonucleotides (PLA probe PLUS and PLA probe MINUS) for 1 hour at 37°C. Afterwards, ligation solution containing 2 oligonucleotides (that hybridize to the PLA probes) and Ligase was added for 30 minutes at 37°C. A closed circle is only formed if the 2 PLA probes are in close proximity. Finally, the closed circle was amplified using rolling-circle amplification reaction and the product was hybridized to fluorescently-labelled oligonucleotides. The fluorescent spots generated from positive interactions were quantified using a Nikon eclipse Ti (Nikon Instruments, Inc) epifluorescence microscope.

Proximity Ligation Assay (PLA) was performed using Duolink In Situ PLA reagents according to the manufacturer’s instructions (Sigma Aldrich). Cultured rat SMC were fixed after treatment with PDGF-BB, rapamycin, or control vehicle with 4% PFA for 15 min and permeabilized with 0.25% Triton X-100 PBS for 15 min at room temperature. Aorta cryosections were post-fixed with pre-cooled acetone for 10 min. The same protocol was then used for cultured cells and cryosections. Incubation with antibodies against H3K4me2 (clone CMA303, Millipore, 05-1338) and TET2 (Abcam, ab124297) was done overnight at 4°C. Cells and sections were incubated with Duolink® In Situ PLA Probe anti-rabbit PLUS (Sigma Aldrich, DUO92002) and anti-mouse MINUS (Sigma Aldrich, DUO92004) secondary antibodies, followed by ligation and amplification with Duolink® In Situ Detection reagents Orange (555nm) (Sigma Aldrich, DUO92007). Finally, staining with ACTA2-FITC antibody (4.4 μg/ml, Clone 1A4, Sigma Aldrich, F3777) was performed on cryosection. Slides were mounted using Duolink® In Situ mounting medium with DAPI (Sigma Aldrich, DUO82040). Images were acquired on a fluorescent microscope (Leica, DMi8) using the Ocular Advanced Scientific Camera Control software (Digital Optics Limited). Image processing and PLA dot quantification was performed using ImageJ.