Effect of RK‐33 on radiation‐induced DNA damage AImmunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2‐Gy radiation and A549 cells pre‐treated with 6 μM RK‐33, 12 h before radiation. Effect of RK‐33 on radiation‐induced DNA damage AImmunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2‐Gy radiation and A549 cells pre‐treated with 6 μM RK‐33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co‐immunofluorescence staining. Scale bar is 2 μm. BA549 cells were pre‐treated with RK‐33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated. CH1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK‐33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times. DH1299 cells, containing a stable non‐homologous end‐joining (NHEJ) reporter construct, were treated with RK‐33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times. EMicroarray results from MDA‐MB‐231 cells treated with RK‐33 and shDDX3 were validated by qRT–PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio‐Rad) and performed in biological triplicates. F, GDNA repair‐related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK‐33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes. Data information: Significance was assessed by two‐sided, paired t‐test. Error bars represent SD. Source data are available online for this figure. Guus M Bol et al. EMBO Mol Med. 2015;7:648-669 © as stated in the article, figure or figure legend