MUS81 and RAD52 promote survival also independently from each other. (A)

<p>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 “<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003910#s4" target="_blank">Materials and Methods</a>” 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>B</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. (<b>C</b>) 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 “<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003910#s4" target="_blank">Materials and Methods</a>” 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. (<b>D</b>) 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. (<b>E</b>) Western blotting showing depletion of protein levels after transfection with the indicated siRNAs. PCNA was used as loading control. (<b>F</b>) 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 “<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003910#s4" target="_blank">Materials and Methods</a>”. 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.