DNA Damage Response in Rhabdomyosarcoma Cells

RH30 and RD cells (5 × 10⁴), seeded onto 2% gelatine coated-glass coverslips in 24-well plates, were allowed to attach overnight and then incubated for 48 h in the presence or absence of Olaparib (5 µM) or AZD2461 (10 µM). For IF analysis of the effects induced by the PARPi and IR combination, RH30 and RD cells, pretreated for 24 h with Olaparib or AZD2461 were irradiated and incubated for additional 4 h at 37 °C. IF assays were performed as previously described (Megiorni et al. 2016 (link)) using the following primary antibodies: Cdc2, p-Cdc2, Cdc25C, Cyclin B1, Cyclin D1, RAD51 (1:20 dilution in PBS; Santa Cruz Biotechnology), and γH2AX (1:500 in PBS; Cell Signaling). All single-stained or merged images were acquired with a Zeiss ApoTome microscope (40× magnification) using the Axiovision software (Carl Zeiss, Jena, Germany). For γH2AX and RAD51, focus fluorescence intensity in the respect of cell number in each analysed field was reported.