Duolink in situ reagents

In situ PLA was performed with DuoLink In Situ Reagents from Olink Bioscience (Sigma-Aldrich). NHK cells were seeded onto glass coverslips coated with rat-tail type I collagen (20 μg ml⁻¹ in PBS, BD Biosciences), grown for 24 h in complete FAD medium, and fixed in 4% (w/v) paraformaldehyde (Sigma) for 10 min at room temperature. PLAs were performed as indicated by the provider's protocol, after an overnight incubation with primary antibodies following the immunofluorescence protocol described above. Primary antibodies are listed in Supplementary Data 13.

The in situ PLA analysis was performed using Duolink^® in situ reagents (O-LINK^® Bioscience, Uppsala, Sweden) according to the manufacturer’s instructions. Cells were transfected with plasmids or siRNAs and then seeded on glass coverslips in 24-well cell culture plates (1 × 10⁵ cells/well). After 24 h growth under standard conditions, cells were treated with EGF (25 ng/mL) in the presence or absence of peptides and each reagent for 15 min in a CO₂ incubator at 37 °C, and then washed twice with 1X PBS. Cells were fixed with 2% formaldehyde in PBS for 10 min at room temperature (RT), and subsequently washed twice with 1X PBS, permeabilized with 0.1% Triton X-100 in PBS, and then washed twice with wash buffer A. Cells were incubated with blocking solution at 37 °C for 30 min, and then washed twice with wash buffer A. Cells were stained with specific antibodies (1:100 for in situ PLA) as indicated. Protein–protein interactions were analyzed using a confocal laser scanning microscope Olympus FluoView FV1000 (Olympus, Tokyo, Japan). PLA signals in cell populations (n = 4) were quantified by NIS-Elements analysis, and four or two independent experiments were performed. The average number of rolling-circle products (RCPs) per cell ± standard error is shown.

In situ PLA was used to evaluate protein interactions. Briefly, cells were seeded in 96-well microtiter plates (0.5–1 × 10⁴/well) and treated as described above. Cells were then washed in PBS, fixed in 4% formaldehyde in PBS, permeabilized with PBS 0.2%Tween-20 (PBS-T) and blocked with 2% BSA in PBS-T. PLA pre-qualified rabbit anti-ErbB2 antibody (Sigma Aldrich) was used together with one of the following primary antibodies, according to the protein interaction analyzed: mouse anti-Cdc37 (Santa Cruz Biot), mouse anti-HSP70 (Sigma Aldrich) or mouse anti-HSP90 (abcam). The PLA reactions were performed using Duolink^® In Situ reagents, from Olink^® Bioscience, according to manufacturer's instructions. Nuclei were stained with Duolink In Situ Microplate Nuclear Stain, Anti-Fade reagent (Sigma Aldrich). Fluorescence images were acquired by the High Content Screening (HCS) system Operetta and analysed with Harmony software.