Biocoat coverslips

For the quantification of MLV titers by coculture, round, 12-mm-diameter BioCoat coverslips (Corning; Fisher Scientific, Merelbeke, Belgium) were first added to a 24-well plate (Nunc; Thermo Scientific, Asse, Belgium). Next, approximately 1.5 × 10⁵ NIH 3T3 cells were seeded into each well. After 24 h, NIH 3T3 cells were cocultured with 10⁴ cells from the spleen or thymus for 24 h. Next, NIH 3T3 cells were rinsed with PBS and fixed with 4% paraformaldehyde. For staining, a primary antibody against the MLV protein p12 was obtained from hybridoma alpha CA cells (ATCC CRL-1890). The primary antibody was incubated for 1 h. Goat anti-mouse biotin (Dako Denmark, Glostrup, Denmark) was used as a secondary antibody, and samples were incubated for 20 min, followed by an incubation step with streptavidin-horseradish peroxidase (HRP) (Dako Denmark) for 30 min. Next, 3,3’-diaminobenzidine (DAB) staining was done. Coverslips were mounted using Mowiol 4-88 (Calbiochem, Merck). Infected cells were counted with a Leica DMR microscope (×40 magnification).

For the quantification of MLV titers by coculture, round, 12-mm-diameter BioCoat coverslips (Corning; Fisher Scientific, Merelbeke, Belgium) were first added to a 24-well plate (Nunc; Thermo Scientific, Asse, Belgium). Next, approximately 1.5 × 10⁵ NIH 3T3 cells were seeded into each well. After 24 h, NIH 3T3 cells were cocultured with 10⁴ cells from the spleen or thymus for 24 h. Next, NIH 3T3 cells were rinsed with PBS and fixed with 4% paraformaldehyde. For staining, a primary antibody against the MLV protein p12 was obtained from hybridoma alpha CA cells (ATCC CRL-1890). The primary antibody was incubated for 1 h. Goat anti-mouse biotin (Dako Denmark, Glostrup, Denmark) was used as a secondary antibody, and samples were incubated for 20 min, followed by an incubation step with streptavidin-horseradish peroxidase (HRP) (Dako Denmark) for 30 min. Next, 3,3’-diaminobenzidine (DAB) staining was done. Coverslips were mounted using Mowiol 4-88 (Calbiochem, Merck). Infected cells were counted with a Leica DMR microscope (×40 magnification).

Cells grown on BioCoat™ coverslips (Corning) were treated with DMSO or 1 µM IQ10 for 4 h prior to 2 Gy irradiation (sham irradiated cells as control). They were then fixed in 4% paraformaldehyde/PBS for 30 min, permeabilised and blocked in 0.3% Triton X-100 with 3% bovine serum albumin in PBS for another 30 min at room temperature. Cells were stained with anti-γH2AX antibody (1:500 dilution, Millipore) overnight at 4 °C followed by incubation with Alexa 647-conjugated goat anti-mouse secondary antibody (1:200 dilution, Abcam) for 1 h. Nuclei were counterstained with VECTASHIELD containing DAPI (Vector). Imaging was performed using a confocal microscope (Leica TCS SPE). The number of γH2AX foci was counted using ImageJ with at least 100 nuclei analysed for each condition.