Fig. 2.

AUY922 reduces HR in response to CCRT but increases 53BP1 focal formation and mutant p53 signaling. a Representative images of RAD51 foci in the HNSCC cell lines CAL27, FaDu and HN5. Nuclear localization indicated by DAPI staining. AUY922 refers to 10 nM added 16 h pre-DNA damage. 2 Gy radiation plus 10 μM cisplatin for brevity is referred to as concurrent-cisplatin radiotherapy (CCRT). b Quantitation of the average RAD51 foci per nucleus at 4 h and for CCRT and CCRT + AUY922 conditions at 24 h also. Values shown are means ± SEM of a minimum of three independent experiments. c Western blot analysis of pS1524 BRCA1 and pS15 p53 signaling post irradiation or cisplatin treatment. d, e, f Automated image based quantification of average nuclear pS1524 BRCA1 intensity, average pS15 p53mt nuclear intensity, and average 53BP1 foci per nucleus with treatment schedule as outlined in panel b. Representative nuclear staining for each cell line and condition are shown. Values shown are means ± SEM from a minimum quantification of 12 fields of view across two independent experiments, except for pS1524 BRCA1 in CAL27 cells which represent 8-10 fields of view from a single experiment. Statistical analysis by 2-tailed t-test; *p < 0.05, **p < 0.01, ***p < 0.001