Fig. 3: AJ-5 activates the DNA damage and the p38 MAPK pathways.

a γH2AX protein levels detected by western blotting in RH30 (aRMS) and RD (eRMS) cells treated with vehicle (V), 0.1 µM or IC₅₀ AJ-5 for 24 and 48 h. p38 was used as a loading control. Densitometry readings were obtained using ImageJ and protein expression levels are represented as a ratio of protein of interest/p38 normalized to the vehicle control sample (where possible). Blots are representative of at least two independent repeats. b Representative confocal immunofluorescence maximum intensity projection images (×630; Carl Zeiss LSM 510) of RH30 and RD cells treated with IC₅₀ AJ-5 or vehicle for 24 h and γH2AX detected with a fluorophore-conjugated Cy3 secondary antibody. Nuclei of cells were stained with DAPI. Scale bar is 20 µM. Box plots represent quantification of γH2AX levels per treatment condition as mean nuclear Cy3 fluorescence from 20 fields of view from three independent repeats. Data were analysed using GraphPad Prism 6.0 and a parametric unpaired t-test was performed where *p < 0.05, **p < 0.01, ***p < 0.001. c Western blot analyses with antibodies to key DNA damage and stress signalling pathway proteins: p-ATM, p-Chk2, p-p38, p53, and p21. RH30 and RD cells were treated with AJ-5 and protein expression quantified as described above in a. Broken lines in western blots shown in this figure indicate where lanes not relevant were removed