1. Tousled‐like kinase 2 regulates recovery from a DNA damage‐induced G2 arrest
U2OS cells were transfected with four independent siRNAs from the pools used in the screen, treated as in Fig , and analyzed for mitotic index. Error bars represent SD, n = 3. Statistical significance was tested using a paired two‐tailed t‐test (NS P > 0.05, *P ≤ 0.05, **P ≤ 0.01). U2OS cells were synchronized with a single thymidine block, released into G2, and damaged with 0.5 μM adriamycin for 1 h. After a 16‐h G2 arrest, cells were harvested for Western blot analysis and analyzed for Tlk2 protein levels. Live cell imaging of thymidine‐synchronized unperturbed or damaged G2 cells. Cumulative percentage of cells entering mitosis were scored and plotted. U2OS cells were transfected with either a control siRNA or Tlk2 siRNA #3, synchronized, and damaged in G2. Cells were either harvested or treated with caffeine for 8 h before harvest, and cell cycle distribution was analyzed by FACS. Percentages of cells in each quadrant are indicated. Tlk2Δ cells were generated using CRISPR/Cas9 genome editing. Cells were synchronized in G2 by thymidine release and damaged with 0.5 μM adriamycin for 1 h. After a 16‐h G2 arrest, cells were induced to recover by addition of caffeine for 8 h and analyzed by FACS. Error bars represent SD, n = 4. Statistical significance was tested using a paired two‐tailed t‐test (NS P > 0.05, *P ≤ 0.05, ***P ≤ 0.001). U2TR cells stably expressing Tlk2 siRNA #3‐insensitive tetracycline‐inducible FLAG‐Tlk2‐wt or FLAG‐Tlk2‐D613A were thymidine‐synchronized and damaged in G2. Tetracycline was present form the start of the experiment where indicated. Caffeine‐induced recovery assay of cell lines shown in (F). Error bars represent SD, n = 3. Statistical significance was tested using a paired two‐tailed t‐test (NS P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).
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EMBO Rep, Volume: 17, Issue: 5, Pages: , First published: 01 March 2016, DOI: ( /embr )