Survival of the Replication Checkpoint Deficient Cells Requires MUS81-RAD52 Function
Figure 6
MUS81 and RAD52 promote survival also independently from each other. (A)
Effect of the down-regulation of RAD52 and/or MUS81 on cell viability. GM01604 cells were transfected with the indicate siRNAs and 48 h later treated with 400 nM UCN-01 and/or 2 mM HU for 6 h. Cell viability was evaluated by LIVE/DEAD assay as described in “Materials and Methods” after 18 h of recovery in HU-free medium, with or without continuous exposure to UCN-01. Data are presented as percentage of dead cells and are mean of three independent experiments. Error bars represent standard error. Where not depicted, standard errors were <15% of the mean. In the panel representative images from samples treated with HU are reported: live cells are green stained while dead cells are red. (B) Analysis of replication checkpoint activation. Cells were treated with 400 nM UCN-01 and/or 2 mM HU for 6 h. Then cells were recovered for 4 h and immunoblotted for pS345CHK1 and CHK1. MUS81 was used to verified protein depletion and Lamin B1 as loading control. The graph shows the gel quantification. (C) Effect of the over-expression of RAD51-T309D on cell viability of cells experiencing replication stress in the absence of MUS81. GM01604 cells were transfected with the indicate siRNAs and 24 h later nucleofected with plasmids expressing either RAD51wt or RAD51-T309D. Twenty-four hours thereafter, cells were treated with 400 nM UCN-01 and/or 2 mM HU for 6 h. Cell viability was evaluated by LIVE/DEAD assay as described in “Materials and Methods” after 18 h of recovery in HU-free medium, with or without continuous exposure to UCN-01. Data are presented as percentage of dead cells and are mean of three independent experiments. Error bars represent standard error. Where not depicted, standard errors were <15% of the mean. (D) Levels of chromatin-bound RAD51 in GM01604 cells transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were treated with UCN-01 for 1 h and then with HU for 6 h. The graph shows the amount of RAD51 in the chromatin fraction determined after densitometry of the representative gels and presented as arbitrary units normalized against the amount of Lamin B1. (E) Western blotting showing depletion of protein levels after transfection with the indicated siRNAs. PCNA was used as loading control. (F) Effect of the down-regulation of RAD51 in RAD52/MUS81-depleted cells on cell viability. GM01604 cells were transfected with control siRNAs (siCtrl) or siRAD52, siRAD51 and siMUS81. Forty-eight hours after interference, cells were treated with 400 nM UCN-01 and/or 2 mM HU for 6 h. Cell viability was evaluated by LIVE/DEAD assay 18 h after recovery in HU-free medium, as described in “Materials and Methods”. Data are presented as percentage of dead cells and are mean of three independent experiments. Error bars represent standard error. Where not depicted, standard errors were <15% of the mean.