ImmunofluorescenceLive cell imaging and microirradiation studies of HeLa cells transfected with peYFP-C1-PALB2 WT or variant constructs were carried out with a Leica TCS SP5 II confocal microscope. To monitor the recruitment of YFP-PALB2 to laser-induced DNA damage sites, cells were microirradiated in the nucleus for 200 ms using a 405-nm ultraviolet (UV) laser and imaged every 30 seconds for 15 minutes. Fluorescence intensity of YFP-PALB2 at DNA damage sites relative to an unirradiated nuclear area was quantified (Supplemental Materials). Cyclin A–positive HeLa cells treated with siCtrl and siRNA against PALB2 were complemented with wild-type and mutant FLAG-tagged PALB2 expression constructs, exposed to 2 Gy of γ-IR, incubated for 6 hours, and subjected to immunofluorescence for RAD51 foci. HeLa cells were fixed with 4% (w/v) paraformaldehyde for 10 minutes at room temperature, washed with tris-buffered saline (TBS), and fixed again with ice-cold methanol for 5 minutes at −20 °C. Cells were incubated for 1 hour at room temperature with the anti-RAD51 (1:7000, B-bridge International, 70-001) and anticyclin A (1:400, BD Biosciences, 611268), and incubated for 1 hour at room temperature with the Alexa Fluor 568 goat antirabbit (Invitrogen, A-11011) and Alexa Fluor 647 goat antimouse (Invitrogen, A-21235) secondary antibodies. Z-stack images were acquired on a Leica CTR 6000 microscope and the number of RAD51 foci per cyclin A–positive cells expressing the indicated YFP-PALB2 constructs was scored with Volocity software v6.0.1 (Perkin–Elmer Improvision). Results represent the mean (± SD) of three independent trials (n = 50 cells per condition). HEK293T cells transfected with PALB2 expression constructs were also subjected to immunofluorescence for PALB2 using the monoclonal anti-FLAG M2 antibody (Sigma) and the Alexa Fluor 568 goat antimouse (Life Technologies) secondary antibody.

RAD51 foci assayHeLa cells were seeded on glass coverslips in 6-well plates at 225 000 cells per well. Knockdown of PALB2 was performed 18 h later with 50 nM PALB2 siRNA using Lipofectamine RNAiMAX (Invitrogen). After 5 h, cells were subjected to double thymidine block. Briefly, cells were treated with 2 mM thymidine for 18 h and release into fresh media for 9 h. Complementation using 800 ng of the peYFP-C1 empty vector or the indicated siRNA-resistant YFP-PALB2 construct was carried out with Lipofectamine 2000 during that release time. Then, cells were treated with 2 mM thymidine for 17 h and protected from light from this point on. After 2 h of release from the second block, cells were irradiated with 2 Gy and processed for immunofluorescence 4 h post-irradiation. Unless otherwise stated, all immunofluorescence dilutions were prepared in PBS and incubations performed at room temperature with intervening washes in PBS. Cell fixation was carried out by incubation with 4% paraformaldehyde for 10 min followed by 100% ice-cold methanol for 5 min at −20°C. This was succeeded by permeabilization in 0.2% Triton X-100 for 5 min and a quenching step using 0.1% sodium borohydride for 5 min. After blocking for 1 h in a solution containing 10% goat serum and 1% BSA, cells were incubated for 1 h with primary antibodies anti-RAD51 (1 :7000, B-bridge International, #70–001) and anti-cyclin A (1:400, BD Biosciences, #611268) diluted in 1% BSA. Secondary antibodies Alexa Fluor 568 goat anti-rabbit (Invitrogen, #A-11011) and Alexa Fluor 647 goat anti-mouse (Invitrogen, #A-21235) were diluted 1:1000 in 1% BSA and applied for 1 h. Nuclei were stained for 10 min with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) prior to mounting onto slides with 90% glycerol containing 1 mg/ml paraphenylenediamine anti-fade reagent. Z-stack images were acquired on a Leica CTR 6000 microscope using a 63× oil immersion objective, then deconvolved and analyzed for RAD51 foci formation with Volocity software v6.0.1 (Perkin-Elmer Improvision). The number of RAD51 foci per cyclin A-positive cells expressing the indicated YFP-PALB2 constructs was scored using automatic spot counting by Volocity software and validated manually. Data from three independent trials (total n = 225 cells per condition) were analyzed for outliers using the ROUT method (Q = 1.0%) in GraphPad Prism v6.0 and the remaining were reported in a scatter dot plot. Intensity values, also provided by Volocity, of 500 RAD51 foci from a representative trial were normalized to the WT mean and reported in a scatter dot plot. Horizontal lines on the plots designate the mean values.

Olaparib sensitivity assayFor the sensitivity assay in HeLa, 240 000 cells were seeded into one well of a six-well plate before being transfected 6–8 h later with 50 nM control or PALB2 siRNA using Lipofectamine RNAiMAX (Invitrogen). The next morning, cells were complemented with 800 ng of the peYFP-C1 empty vector or the indicated siRNA-resistant YFP-tagged PALB2 construct using Lipofectamine 2000 (Invitrogen) for 24 h and then seeded in triplicates into a Corning 3603 black-sided clear bottom 96-well microplate at a density of 3000 cells per well. The remaining cells were kept and stored at −80°C until processed for protein extraction and immunoblotting as described above. Once attached to the plate, cells were exposed to different concentrations of olaparib (Selleckchem, #S1060) ranging from 0 (DMSO) to 2.5 μM. After 3 days of treatment, nuclei were stained with Hoechst 33342 (Invitrogen) at 10 μg/ml in media for 45 min at 37°C. Images of entire wells were acquired at 4x with a Cytation 5 Cell Imaging Multi-Mode Reader followed by quantification of Hoechst-stained nuclei with the Gen5 Data Analysis Software v3.03 (BioTek Instruments). Cell viability was expressed as percentage of survival in olaparib-treated cells relative to vehicle (DMSO)-treated cells. Results represent the mean ± SD of at least 3 independent experiments, each performed in triplicate.

To further assess the impact of the 5 selected VUS on PALB2, we examined whether they affected the accumulation of RAD51 at IR-induced DSBs by measuring the formation RAD51 foci.