Analysis of replication stress by BrdU native detection assay

For detection of long fragments of single-stranded DNA (ssDNA), a feature of replication stress, we grow keratinocytes in coverslips [induced with 50 nM 4OHT, noninduced (control), treated and nontreated with 5 nM NAC] in the presence of 50 mM 5-bromo-2’-deoxyuridine (BrdU) for 24 h to allow its incorporation into DNA. After that, we washed the coverslip-containing cells using PBS and fixed them using 4% of paraformaldehyde diluted in PBS for 10 min at room temperature. Next, cells were washed with PBS and permeabilized with 0.2% Triton-X 100 for 10 min at room temperature. To ensure that all cells incorporated BrdU, replicates of each condition were subjected to DNA denaturation using 2 M HCl. Then, all samples were washed, and BrdU was detected (when accessible) using α-BrdU-rat (Abcam) for 3 h at room temperature, followed by a 3-h incubation with the secondary antibody Alexa Fluor 555-conjugated goat anti-rat (Thermo Scientific). After that, the coverslip-containing cells were washed using 1× PBS and deposited on slides. VECTASHIELD^® Mounting Medium with DAPI (Vector Labs) was used to be an antifade mounting solution and to stain nuclear DNA.