Fig. 2

Etoposide activates DDR in MCL cells. a. NCEB1, JeKo1 and REC1 cells were treated with etoposide (4 μg/ml) or DMSO, as a control, and harvested 3, 6 or 12 h later. Cells were then processed for ICF with anti-γH2AX Ab, counterstained with DAPI and analyzed by confocal microscopy. b. MCL cells were treated with etoposide (4 μg/ml) or vehicle (0). Whole cell proteins were purified, 40 μg of proteins were loaded on gels, separated by SDS-PAGE, transferred onto nitrocellulose membranes and incubated with anti-γH2AX Ab. An anti-β-actin Ab served as a control for gel loading and transfer. c. MCL cells were treated with 4 μg/ml etoposide for 24 h and harvested. Western blots were performed as in B. Membranes were incubated with Abs anti-pT68-CHK2, −CHK2 and -β-actin (upper part). The density of specific bands was measured by densitometry and the ratio pCHK2/CKH2 indicated under the blots. Lower part, membranes were incubated with anti-pS15-p53, −p53, −p21 and -β-actin Abs as already described. d. Cultured REC1 and NCEB1 cells were treated as in b and western blots were done as described using Abs anti-pT68-CHK2 and -β-actin