Supplementary Material for: Compromised DNA Repair and Signalling in Human Granulocytes Ponath V. Heylmann D. Haak T. Woods K. Becker H. Kaina B. 10.6084/m9.figshare.7178315.v1 https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Compromised_DNA_Repair_and_Signalling_in_Human_Granulocytes/7178315 In previous studies, we showed impaired DNA repair in human monocytes. Here, we addressed the question of whether human neutrophilic granulocytes that arise from the same precursor as monocytes exhibit a similar phenotype and are impaired in repairing their DNA. We show that neutrophilic granulocytes isolated from peripheral blood display a lack of the same repair proteins that are missing in monocytes and do not show repair of their DNA when damaged by ionising radiation (IR) or chemical ROS. Contrary to T cells, we observed no decline in the number of single-strand breaks following γ-radiation. Also, granulocytes did not show γH2AX foci formation while T cells and peripheral blood lymphocytes (PBL) responded. In comparison to PBL, XRCC1, PARP-1 and ligase III were not expressed and there was also no discernible signal for key damage response proteins ATM, ATR and DNA-PK<sub>CS</sub> as well as γH2AX in neutrophils. Time course and dose-response experiments confirmed the absence of H2AX phosphorylation after radiation treatment although an accumulation of double-strand breaks was detected in the neutral Comet assay. Overall, the data indicate that terminally differentiated neutrophilic granulocytes in the peripheral blood display strong downregulation of DNA repair and DNA damage response factors, which should be taken into account if studies with whole peripheral blood containing granulocytes are performed, causing a significant intra-experimental variation in the cellular repair capacity. 2018-10-08 13:32:13 DNA repair Neutrophilic granulocytes DNA damage Cell death Immune cells