DNA Damage Response Pathway Knockdown

The Human ON-TARGETplus siRNA Library-DNA Damage Response and the siRNA targeting OGG1, PARG, Pol β, and XRCC1, each containing a pool of four different siRNA sequences to help aid knockdown efficiencies, were from Horizon Discovery (Cambridge, UK). The non-targeting control siRNA (AllStars Negative Control siRNA) was from Qiagen (Manchester, UK). The following antibodies were used: γH2AX (05–636; Merck-Millipore, Watford, UK), OGG1, PARG, phosphorylated ATM and phosphorylated DNA-PKcs (ab124741, ab169639, ab81292 and ab18192; Abcam, Cambridge, UK), PARP-1 (sc-53643; Santa Cruz Biotechnology, Heidelberg, Germany), OGG1 for immunofluorescence (NB100-106; Bio-Techne Ltd, Abingdon, UK), Polβ and XRCC1 (kindly provided by G. Dianov) and tubulin (T6199; Sigma-Aldrich, Gillingham, UK). Goat anti-mouse Alexa Fluor 555 or goat anti-rabbit Alexa Fluor 488 secondary antibodies for immunofluorescence were from Life Technologies (Paisley, UK). The OGG1 inhibitor TH5487, as previously described [21 (link)], was used at a concentration of 10 µM. The PARG inhibitor PDD00017273, also previously described [22 (link)], was used at a concentration of 1 µM.