Fig. 5
PARP1 and PARP2 are required to stabilise Rad51 at stalled and/or damaged replication forks. a The indicated cell lines were treated with 1 μM EdU (Unt), or with 1 μM EdU and 0.5 mM MMS for 1 h before recovery in fresh media, and RPA foci quantified in EdU-positive cells. Times indicated show hours since addition of EdU and MMS. Representative images of EdU-positive nuclei 12 h after addition of MMS are shown (upper panel). Scale bars represent 10 μm. Error bars represent the SEM from three independent experiments. b The indicated cell lines transfected with control or XRCC1 siRNA were treated as in a and RPA nuclear foci analysed in EdU-positive cells. Cells were fixed 12 h after addition of EdU and MMS. Error bars represent the SEM from three independent experiments. c The indicated cell lines, transfected with control or XRCC1 siRNA, were left untreated (–) or exposed to 0.5 mM MMS for 1 h before recovery in fresh media for 12 h (+). Western blotting of whole cell extracts was performed with the indicated antibodies. d U2OS cells transfected with control or Fbh1 siRNA –/ + olaparib (PARPi), were treated as in a and Rad51 nuclear foci analysed in EdU-positive cells. Cells were fixed 12 h after addition of EdU and MMS. Knockdown of Fbh1 was confirmed by western blotting. Error bars represent the SEM from three independent experiments. e U2OS or parp1/2Δ cells transfected with control or Fbh1 siRNA were treated as in a and Rad51 nuclear foci analysed in EdU-positive cells. Times indicated show hours since addition of EdU and MMS. Representative images of EdU-positive nuclei 12 h after MMS addition are shown. Scale bars represent 10 μm. Error bars represent the SEM from three independent experiments. f Flp-In T-Rex HT1080 cells expressing FE-PALB2, with or without Olaparib (PARPi), were left untreated (–) or exposed to 0.5 mM MMS for 1 h before recovery in fresh media (+). Extracts were prepared 6 h after MMS addition. Western blotting of whole cell and chromatin extracts was performed with the indicated antibodies. In all cases, statistical significance was determined using a two-tailed Student’s t-test (NS, not significant; *** p < 0.001)
