Goat serum

After reprogramming, the iPSCs (passage 10) were cultured on coverslips and fixed with 100 % methanol (20′, −20 °C). Then, they were permeabilized using 0.1 % Triton X-100 solution (Sigma-Aldrich) (15′, room temperature (RT)). Non-specific binding was blocked using 5 % goat serum (Jackson ImmunoResearch) (30′, RT) and the primary antibodies were added and incubated overnight (4 °C). Hereafter, the cells were washed using 0.1 % Triton X-100 and secondary antibodies were incubated for one hour (RT). DAPI (Life Technologies) was used to visualize the cell nuclei. Coverslips were mounted on glass slides and pictures were taken using a 20x objective from Olympus BX51 fluorescence microscope.

IR-induced 53BP1 kinetics were determined as previously outlined with modifications (19 (link)). Cells were grown on coverslips one day before the experiment and on the day of the experiment, cells were exposed to 1Gy of γ-rays. At different time points after IR (see figure), cells were washed twice with ice cold 1× PBS and fixed with 4% paraformaldehyde (in 1× PBS) for 20 min at RT, washed 5 times with 1× PBS, and incubated in 0.5% Triton X-100 on ice for 10 min. Cells were washed 5 times with 1× PBS and incubated in blocking solution (5% goat serum (Jackson Immuno Research) in 1× PBS) overnight. The blocking solution was then replaced with the 53BP1 (SC-22760, Santa Cruz) primary antibody diluted in 5% goat serum in 1x PBS and the cells were incubated for 2 h. Cells were then washed 5 times with wash buffer (1% BSA in 1× PBS). Cells were incubated with the Alexa Fluor 488 (1:1000) (Molecular Probes) secondary antibody in 1% BSA, 2.5% goat serum in 1× PBS for 1 h in the dark, followed by five washes. After the last wash, cells were mounted in VectaShield mounting medium containing DAPI. The images were acquired using a Zeiss Axio Imager fluorescence microscope utilizing a 63× oil objective. ≥100 cells were analyzed for each time point.

Microneutralization assays of SARS-CoV-2 virus were performed as previously described^{3 (link)}. The day before infection, Vero E6 cells were seeded at 1 × 10⁴ cells per well into 96-well plates. The diluted plasma and antibodies were mixed with SARS-CoV-2 WA1/2020 or the B.1.351 variant and incubated for 1 h at 37 °C. The antibody–virus mix was then directly applied to Vero E6 cells and incubated for 22 h at 37 °C. Cells were subsequently fixed by adding an equal volume of 70% formaldehyde to the wells, followed by permeabilization with 1% Triton X-100 for 10 min. After washing, cells were incubated for 1 h at 37 °C with blocking solution of 5% goat serum in PBS (catalogue no. 005–000-121; Jackson ImmunoResearch). A rabbit polyclonal anti-SARS-CoV-2 nucleocapsid antibody (catalogue no. GTX135357; GeneTex) was added to the cells at 1:1,000 dilution in blocking solution and incubated at 4 °C overnight. Goat anti-rabbit AlexaFluor 594 (catalogue no. A-11012; Life Technologies) was used as a secondary antibody at a dilution of 1:2,000. Nuclei were stained with Hoechst 33342 (catalogue no. 62249; Thermo Fisher Scientific) at 1 μg ml⁻¹. Images were acquired with a fluorescence microscope and analysed using ImageXpress Micro XLS (Molecular Devices). All experiments were performed in a biosafety level 3 laboratory.

The paraffin sections were subjected to gradient ethanol dewaxing and followed by antigen repair. Then, the samples were blocked with 10% goat serum (#005-000-121, Jackson Immuno-Research, USA) for 1 h at room temperature, and incubated overnight with primary antibodies at 4 °C: antibodies against GPX4 and GCLM were used at 1:100 dilution. The next day, the samples were incubated with secondary antibodies for 1 h at 37 °C: HRP-conjugated goat anti-rabbit IgG (used at 1:200 dilution, ABclonal). Images were acquired by microscopy.