Alexaflour 546

Antibodies against TOP1, IκBα, phospho‐ATM, γH2AX, DNA. FLAG, β‐actin, Anti‐IKKγ/NEMO TDP1 (#SAB1411073), and H2AX were purchased from Sigma (St. Louis, MO). Antibodies against RPA2 and phospho‐RPA2 were from Bethyl Laboratories (Montgomery, TX). Antibodies against p65, WRN (#SC5629), ATM (#SC23921), CHK1 (#SC8404), Lamin B (#SC6216), and CRISPR‐Cas9 double nickase plasmid (control and WRN) were from Santa Cruz biotechnology (Santa Cruz, CA). Anti‐phospho‐345‐CHK1 was from Epitomics (Cambridge, UK). Anti‐BrdU (#347580), anti‐PAR from BD Biosciences (San Jose, CA). Anti MRE11 (#4847) from Cell Signaling Technology (Massachusetts, USA). AlexaFlour‐546 and AlexaFlour‐488 tagged secondary antibodies from Jackson ImmunoResearch, (PA, USA). Lipofectamine 2000/3000, Alexa Flour‐488/555/595 (#A21123/#A31570), prolong anti‐fade Gold was obtained from (Life Technologies, Carlsbad, CA). SCH900776 (CHK1 inhibitor) was purchased from Selleckchem (Houston, USA). Talazoparib (BMN673; PARP inhibitor) was procured from ApexBio, USA. All other reagents like Ro106‐9920, mirin (MRE11 inhibitor), camptothecin, EdU, etc., were obtained from Sigma Chemicals (St. Louis, MO), unless mentioned in the respective places.

Cells were fixed with 4% paraformaldehyde for 10 min and blocked with skimmed milk in PBST (0.1% Triton X-100 in PBS) for 1 h. Cells were then incubated with rabbit anti-SLD5 (1:200) or mouse anti-Ki-67 (1:200) overnight at 4 °C, washed with PBS, and then incubated with donkey anti-rabbit Alexa Fluor 488 (1:200, Jackson ImmunoResearch) or goat anti-mouse Alexa Flour546 (1:200, Jackson ImmunoResearch) for 1 h at room temperature. Nuclear DNA was counterstained with DAPI (1:3000 Dojindo). Stained samples were assessed under a microscope and images were analyzed using ImageJ software.