Figure 5. RPA phosphorylation... | Rockefeller University Press

Figure 5.

$Figure 5. RPA phosphorylation facilitates proper nuclear localization of both PALB2 and BRCA2. (A) RPA and PALB2 show significant colocalization during replication stress. U2-OS cells, transiently transfected with a GFP-PALB2 expression vector, were mock treated or treated with 1 mM HU for 3 h. Cells were then detergent extracted to remove the soluble fraction of the GFP-PALB2 and RPA pools and fixed. After staining for RPA2, cells were imaged for GFP-PALB2, RPA2, and DAPI. (B and C) Mutation of RPA2 phosphorylation sites reduces GFP-PALB2 nuclear retention during replication stress. Cells in which endogenous RPA2 was replaced with ectopic WT-, PIKK_A, or Cdk_A-RPA2 were transfected with a GFP-PALB2 expression vector. Cells were treated with 1 mM HU for 3 h and then imaged as described in A. Images were quantitated using ImageJ (National Institutes of Health), and the relative amount (rel. amt) of PALB2 nuclear staining is shown. Error bars indicate SDs. (D) Mobility of PALB2 under replication stress conditions is significantly higher in cells replaced with either the Cdk_A- or PIKK_A-RPA2 mutant. FRAP analysis of PALB2 mobility is shown, with the data corrected for acquisition bleaching. Prebleach data were used to determine SDs. The calculated t1/2 of recovery is shown below the plot. Each combination of RPA2 variant and condition tested was repeated five to seven times. (E) Mutation of RPA phosphorylation sites causes loss of BRCA2–RPA colocalization during replication stress. U2-OS cells were treated with 1 mM HU for 3 h and then extracted and fixed. Cells were stained for BRCA2, RPA2, and DAPI and imaged. (F) PALB2 and BRCA2 are each in close proximity to RPA in cells, particularly after replication stress. U2-OS cells, either transfected with GFP-PALB2 (two left images) or not transfected (two right images), were either mock treated or incubated with 1 mM HU for 3 h. Cells were analyzed for proximal association of GFP-PALB2 and RPA, or BRCA2 and RPA, using the Duolink immunoassay. In brief, the Duolink assay involves two different DNA-conjugated secondary antibodies that, when adjacent to each other, support formation of a circular DNA molecule that can be amplified by a rolling circle DNA replication. The DNA product is then subsequently detected with a complementary and fluorescently labeled oligonucleotide (Söderberg et al., 2006), giving the observed red dots. Bars: (A) 10 µm; (B, E, and F) 15 µm.$

RPA phosphorylation facilitates proper nuclear localization of both PALB2 and BRCA2. (A) RPA and PALB2 show significant colocalization during replication stress. U2-OS cells, transiently transfected with a GFP-PALB2 expression vector, were mock treated or treated with 1 mM HU for 3 h. Cells were then detergent extracted to remove the soluble fraction of the GFP-PALB2 and RPA pools and fixed. After staining for RPA2, cells were imaged for GFP-PALB2, RPA2, and DAPI. (B and C) Mutation of RPA2 phosphorylation sites reduces GFP-PALB2 nuclear retention during replication stress. Cells in which endogenous RPA2 was replaced with ectopic WT-, PIKK_A, or Cdk_A-RPA2 were transfected with a GFP-PALB2 expression vector. Cells were treated with 1 mM HU for 3 h and then imaged as described in A. Images were quantitated using ImageJ (National Institutes of Health), and the relative amount (rel. amt) of PALB2 nuclear staining is shown. Error bars indicate SDs. (D) Mobility of PALB2 under replication stress conditions is significantly higher in cells replaced with either the Cdk_A- or PIKK_A-RPA2 mutant. FRAP analysis of PALB2 mobility is shown, with the data corrected for acquisition bleaching. Prebleach data were used to determine SDs. The calculated t_1/2 of recovery is shown below the plot. Each combination of RPA2 variant and condition tested was repeated five to seven times. (E) Mutation of RPA phosphorylation sites causes loss of BRCA2–RPA colocalization during replication stress. U2-OS cells were treated with 1 mM HU for 3 h and then extracted and fixed. Cells were stained for BRCA2, RPA2, and DAPI and imaged. (F) PALB2 and BRCA2 are each in close proximity to RPA in cells, particularly after replication stress. U2-OS cells, either transfected with GFP-PALB2 (two left images) or not transfected (two right images), were either mock treated or incubated with 1 mM HU for 3 h. Cells were analyzed for proximal association of GFP-PALB2 and RPA, or BRCA2 and RPA, using the Duolink immunoassay. In brief, the Duolink assay involves two different DNA-conjugated secondary antibodies that, when adjacent to each other, support formation of a circular DNA molecule that can be amplified by a rolling circle DNA replication. The DNA product is then subsequently detected with a complementary and fluorescently labeled oligonucleotide (Söderberg et al., 2006), giving the observed red dots. Bars: (A) 10 µm; (B, E, and F) 15 µm.