Figure 6. LIN37 prevents resection and HR through suppressing HR protein expression in non-cycling cells.

(A) Western blot analysis of proliferating Lig4^−/−:Trp53bp1^−/− or Lig4^−/−:Lin37^−/− abl pre-B cells with or without indicated gRNAs following Cas9 induction for bulk gene inactivation using the indicated antibodies (left). Flow cytometric analysis of chromatin-bound RPA before and after IR of the same cells after rendered non-cycling by imatinib treatment (right). Representative of three experiments. (B) Quantification of RAD51 foci in non-cycling Lig4^−/−, Lig4^−/−:Trp53bp1^−/−, and Lig4^−/−:Lin37^−/− abl pre-B cells before IR treatment and 4 and 20 hr after IR. Red bars indicate the median number of RAD51 foci in each sample of more than 1000 cells analyzed for each cell line. Representative of two independent experiments (****p<0.0001, Mann-Whitney test). (C) Flow cytometric analysis of HR-mediated DSB repair in non-cycling Lig4^−/− and Lig4^−/−:Lin37^−/− abl pre-B cells using the HPRT-DR-GFP reporter. The percentage of GFP-positive cells is shown. Error bars are ± SEM from three experiments (*p=0.0124, t-test). DSB, double-strand break; gRNA, guide RNA; HR, homologous recombination; IR, ionizing radiation.

Figure 6—figure supplement 1. LIN37 deficiency leads to RAD51 focus formation in non-cycling abl pre-B cells.

Representative images of RAD51 IR-induced foci in non-cycling Lig4^−/−, Lig4^−/−:Trp53bp1^−/−, and Lig4^−/−:Lin37^−/− abl pre-B cells. IR, ionizing radiation.