Duolink 2 pla probes

Cells were fixed for 20 min with 4% paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized for 15 min with 0.2% Triton X-100 in PBS. An in situ PLA for detection of EGFR homodimers was performed with Duolink II PLA probes, Probemaker, and detection reagents (Olink Bioscience). Rabbit monoclonal antibodies to EGFR (Abcam) were conjugated to PLUS or MINUS PLA oligonucleotide arms with the use of Probemaker. Cells grown on cover glasses or tumor sections were incubated overnight at 4°C with the antibody-oligonucleotide complexes (PLA probes). Annealing of the PLUS and MINUS PLA probes occurs when two EGFR monomers are in close proximity, and repeat sequences in the annealed oligonucleotide complexes are amplified and then recognized by a fluorescently labeled oligonucleotide probe. PLA signals were detected with a Keyence BZ-8100 fluorescence microscope and were quantified with BZ Analyzer software (Keyence); 20 cells for each cell line and all cells in each image for tumor sections were examined for determination of the number of PLA signals per cell.

Cells were grown (and transfected in the case of U2OS cells) as indicated on Poly-L-Lysine-coated microscope slides (Sigma). Cells were fixed in PBS containing 4% Paraformaldehyde (Merck, Nottingham, United Kingdom) for 15 minutes and permeabilized with PBS containing 0.25% Triton X-100 (Sigma) for 10 minutes. Slides were blocked in PBS containing 0.05% Tween and 10% normal donkey serum (Jackson Immunoresearch), and incubated with indicated antibodies against SIRT1 (D739, #2493) (Cell Signaling) and FLAG (M2, F1804) (Sigma), (in transfected U2OS cells), or SIRT1 (D739, #2493) (Cell Signaling) and FOXA2 (M-20, sc-6554) (Santa Cruz Biotechnology) (in HepG2 cells). Cells were subsequently incubated with Duolink II PLA probes (Olink Biosciences) and stained according to manufacturer's protocol. Cells were analyzed with a 63× objective on a Zeiss LSM 710 confocal microscope (Oberkochen, Germany). In experiments in which HepG2 cells were starved, cells were washed thrice with PBS. Subsequently, cells were cultured for 2 or 4 hours in glucose-free medium (Gibco) without FCS prior to fixation.