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. 2019 May 2;47(12):6299–6314. doi: 10.1093/nar/gkz309

Figure 4.

Figure 4.

Active RNAPII is required for HR repair at the G1 phase. (A) To recover after DNA damage breaks, U2OS cells were treated with phleomycin (10 μg/ml) for 1 h, and then phleomycin-removed cells were maintained into new medium for 12 h. Fixed cells were immunostained with anti-pY142 and anti-γH2AX antibodies as well as 5-EU. Scale bars, 10 μm. (B) U2OS cells were transfected with control siRNA or RNAPII-targeting siRNA for 48 h. Micro-irradiated cells were immunostained with anti-RNAPII and anti-γH2AX antibodies as well as 5-EU after 2 h. Scale bars, 10 μm. Data represent mean ± SEM of more than 20 cells. (C) Comparative analysis of HR repair by depletion or inhibition of RNAPII in asynchronous or G1-arrested HR reporter cells. ATM inhibitor (ATMi) or flavopiridol (FP) was used for efficiency of HR repair or of transcription inhibition, respectively. Data represent mean ± SEM of three independent experiments. n.s. means not significant. (D) U2OS cells were transfected with control siRNA or RNAPII-targeting siRNA for 48 h. Micro-irradiated cells were immunostained with indicated antibodies and 5-EU after 2 h. Flavopiridol (FP) was treated before 1 h microirradiation. Scale bars, 10 μm. Data represent mean ± SEM of three independent experiments. n.s. means not significant. (E and F) HeLa cells were transfected with RNAPII-targeting siRNA and arrested at G1 phase by double thymidine block. Flavopiridol (FP) was treated before 1 h microirradiation (E) or RNase A was treated after 2 h microirradiation (F). Fixed cells at 2 h post-microirradiation were immunostained with indicated antibodies. An anti-Cyclin E antibody was used for G1-specific. Scale bars, 10 μm. Data represent mean ± SEM of three independent experiments. n.s. means not significant. (G) 293T cells were transfected with RNAPII 3′-UTR-targeting siRNA (3′UTR) or siRNAPII (3′UTR) and ectopic FLAG-RNAPII constructs (WT or ΔCTD) for 48 h, then cells were irradiated with IR at the indicated dose. The viability was performed by colony forming assay. Data represent mean ± SEM of three independent experiments.