Figure 3.

POLQ suppresses DSB formation upon replication stress. A, U2OS cells with or without POLQ shRNA-1 expression were either treated with HU (2 mm, 24 h) or not and were lysed for Western blot analysis of γH2AX, using Ku70 as the loading control. γH2AX levels were quantified and normalized to the loading control (Ku70), and relative γH2AX levels are shown on the left, with γH2AX levels in the sample post-HU treatment without POLQ shRNA-1 set at 1. The efficiency of POLQ knockdown is shown in Fig. 1C (right). B, POLQWT and POLQKO-1 U2OS cells were infected with lentiviruses encoding ATR shRNA (shATR) or control, and cell lysates were prepared for Western blot analysis of the indicated proteins. The relative levels of γH2AX are shown by quantifying the band density and normalizing to Ku70, with the WT sample expressing ATR shRNA set as 1. C, comet assays were performed with control and POLQKO-1 U2OS cells after infection with lentiviruses encoding ATR-shRNA or vector. Left, images of the comet assays are shown. The length of the tail moment is plotted using dots (right), and tail moments were exported as box-and-whisker plots. The 25th to 75th percentiles and the median are shown in each box. The whisker ends show the minimum and maximum values. Data were analyzed by one-way analysis of variance, and the p value is shown (**, p < 0.01). In all experiments, error bars represent the S.D. of at least three independent experiments.