Survival of the Replication Checkpoint Deficient Cells Requires MUS81-RAD52 Function
Figure 1
MUS81 promotes DSB formation and cell viability in response to replication checkpoint down-regulation.
(A) Analysis of protein depletion by Western blotting in GM01604 cells after transfection with control siRNAs directed against GFP (siCtrl) or siCHK1, siATR, siTOPBP1, siRAD9, siTIPIN and siCHK2, alone or in combination with siMUS81. Immunoblotting was assessed 48 h after transfection using the appropriate antibodies. PCNA was used as loading control. (B) DSBs accumulation by neutral Comet assay in GM01604 cells transfected as in (A) and treated with 2 mM HU for 6 h before subjecting to Comet assay. Graph shows data presented as mean tail moment +/− SE from three independent experiments. Error bars represent standard errors. Where not depicted, standard errors were <15% of the mean. In the panel representative images from selected samples are shown. (C) Evaluation of cell death after replication stress in GM01604 cells transfected with control siRNAs (siCtrl) or siCHK1, siATR and siTIPIN alone or in combination with siMUS81. Forty-eight hours after RNAi, CHK1 inhibitor (UCN-01), ATR inhibitor (ETP-46464) or solvent (DMSO) was added to media 1 h prior HU treatment. After 6 h of HU, cells were recovered overnight before being analysed. Cell viability was evaluated by LIVE/DEAD assay as described in “Materials and Methods”. Data are presented as percentage of dead cells and are mean values from three independent experiments. Error bars represent standard error. Where not depicted, standard errors were <15% of the mean. The panel shows representative images: live cells are green stained, while dead cells are red.