Fig 1: FBXL10, RNF68, and RNF2 are recruited to sites of DNA damage in a PARP1- and TIMELESS- dependent manner.(A) Super-resolution imaging was performed in U-2OS cells to analyze the colocalization between XRCC5 (magenta) and FBXL10, RNF68 and RNF2 (green), with or without 20 min of NCS treatment. Representative nuclei are shown. A white dash line denotes the border of each nucleus. Scale bar represents 2.5 µm. The signal overlap (white) demonstrates the colocalization between XRCC5 and FBXL10, RNF68 or RNF2. (B) The graphs display the correlation between XRCC5 and FBXL10 (NCS-, n = 113; NCS+, n = 109), RNF68 (NCS-, n = 254; NCS+, n = 232) or RNF2 (NCS-, n = 128; NCS+, n = 105) in individual nuclei, respectively. Each plot is the correlation amplitude between XRCC5 and FBXL10/RNF68/RNF2 within one nucleus. The n value representing the total number of analyzed nuclei is pooled from the three or four independent experiments. A.U., arbitrary units. Whisker box shows mean and S.D. values. P values were calculated using two-sample t-test between NCS - and NCS + samples. (C) U-2OS cells stably expressing GFP-FBXL10 (left), mCherry-RNF68 (middle) or GFP-RNF2 (right) were transfected with siRNAs targeting PARP1, TIMELESS, or a non-targeting control (CTRL). Cells were pre-sensitized with BrdU (10 µM) for 36 hr and subjected to 405 nm laser induced damage. Where indicated, cells were pretreated with 1 µM PARP inhibitor (Olaparib) for 1 hr. DNA damage recruitment dynamics were captured by live cell imaging. Relative fluorescence values and images were acquired every 5 s for 4 min. For each condition, =25 cells were evaluated from 2 or three independent experiments. Mean relative fluorescence values and standard errors were plotted against time. Representative images are shown in Figure 1—figure supplement 2A. Times are indicated in seconds. The efficiency of PARP1 and TIMELESS depletion is shown using immunoblotting.