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. 2016 Apr 4;17(5):671–681. doi: 10.15252/embr.201541455

Figure 3. PALB2 phosphorylation promotes RAD51 foci formation.

Figure 3

  1. PALB2 cell lines transfected with PALB2 siRNA followed by inducible expression of siRNA‐resistant PALB2 versions were exposed to 15 Gy of IR and fixed for IF 2 h later. Z‐stack images were acquired with a 60× oil objective (Deltavision). Z‐stack max intensity projection, background subtraction, and foci count were done using ImageJ/Fiji (*P < 0.0001, unpaired Student's t‐test). Scale bar = 5 μm.
  2. PALB2 cell lines were transfected and the exogenous PALB2 induced as in Fig 3A before they were pulsed with 10 μM EdU for 20 min prior to addition of 2 mM HU. Twenty‐four hours later, the cells were fixed and processed for IF as in (A). Cells in S phase (EdU+) at the time of HU treatment were Click‐IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU‐positive cells were enumerated using ImageJ/Fiji (*P < 0.0001, unpaired Student's t‐test). Scale bar = 5 μm.
  3. HeLa DR‐GFP cell line was used to analyze homologous recombination activity. Cells were transfected with PALB2 siRNA followed by transfection of I‐SceI together with empty vector (EV), WT, TMA‐, or TMD‐PALB2 the next day. 72 h later cells were fixed and analyzed by FACS. The percentage of cells with GFP signal from the empty vector sample was set to one (dotted line). The graph shows a representative image from three biological repeats (error bars = SEM).
  4. Phosphorylation of PALB2 supports RAD51 foci. Cells were transfected with siPALB2 overnight followed by forward transfection of siFBH1/siBLM for 6 h then induced for expression of PALB2 (WT and TMA). 24 h following induction cells were pulsed with EdU and exposed to HU as in (B). Cells in S phase (EdU+) at the time of HU treatment were Click‐IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU‐positive cells were enumerated using ImageJ/Fiji (*P < 0.0001, unpaired Student's t‐test). Representative images displaying RAD51 (green) and HA‐PALB2 (red) localization in HU‐treated EdU‐positive (not shown) cells. Scale bar = 5 μm.