Duolink proximity ligation assay

To analyse receptor dimerisation and dimer localisation, Duolink proximity ligation assay (Sigma-Aldrich) was used according to the manufacturer’s instructions. Briefly, cells were stimulated for 72 h with soraphen A and starved 24 h before analysing. To evaluate EGFR-HER2 dimerisation, cells were fixed with methanol for 10 min at −20 °C, washed three times and blocked with 2% BSA for 10 min at RT. Primary antibodies were diluted 1 : 100 in blocking solution and added to the cells. After three washing steps the samples were incubated with PLA probes for 1 h at 37 °C. Ligation was performed at 37 °C for 30 min and the amplification reaction was conducted at 37 °C for 100 min. Before fixation the nuclei were stained with Hoechst33342 (Sigma-Aldrich). Receptor dimerisation was analysed by confocal microscopy.

Proximity ligation assay (PLA) was performed with Duolink® Proximity Ligation Assay (Sigma-Aldrich-Merck, DUO92102) according to manufacturer’s instructions. In brief, 1 × 10⁴ U2OS cells were seeded on glass coverslip and after 12 h were either left untreated, or DSBs were induced with 5 Gy ionizing radiation with a Gammacell 40 Exactor (Best Theratronics). Cells were then fixed, permeabilized as in the immunofluorescence protocol above. Blocking and incubation with primary antibodies against γH2AX (Millipore, 05–636) and THRAP3 (Novus Biologicals, NB100-40848) were performed overnight in a humidified chamber. PLA mouse and rabbit probes were added and ligated, before rolling circle amplification according to the manufacturer’s instructions. Slide preparation and imaging acquisition were performed as in the immufluorescence protocol above. DAPI staining was used to count nuclei and for defining nuclei boundaries as ROIs. PLA products per nucleus were counted with ImageJ using in-house developed Java Macro after background subtraction with rolling ball radius of 10 pixels. The number of colocalization events is reported in boxplot for untreated and IR conditions. Significance was calculated with one-way ANOVA statistics, and *** corresponds to a P-value lower than 0.0001.

NIH/3T3 cells were maintained in DMEM supplemented with 10% FBS, 2 mM glutamine, and 1% penicillin-streptomycin (Invitrogen). Cells were plated on 6-well tissue culture plates (Falcon) and allowed to proliferate until an approximate density of 50% confluence was obtained before transfection. Transfections were performed using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). A total of 5 µg plasmid DNA was added per well, using a plasmid encoding EGFP to keep the total amount of DNA constant between treatments. The plasmid encoding p75 was provided by Phil Barker (University of British Columbia-Okanagan, Kelowna, British Columbia, Canada). For proximity ligation assays, the DuoLink Proximity Ligation Assay (Sigma-Aldrich) was used according to the manufacturer’s instructions. NIH/3T3 cells were transfected with p75 and Ret51, or p75 and HA-tagged Ret51. Antibodies used for proximity ligation assays were p75 (1:500; Advanced Targeting Systems) and HA (1:500; Sigma-Aldrich), with DuoLink In Situ Orange Mouse/Rabbit kit used for secondary antibody and amplification. DAPI was used for nuclear localization.