Fig. 5: Binding to SART3 is required for optimal DDX1 accumulation at sites of damage.

a HEK293T cells transfected with the indicated constructs were lysed in the presence of RNase A, followed by immunoprecipitation and immunoblotting. b U2OS cells stably expressing GFP or GFP-SART3 were transfected with siNC or siSART3, followed by treatment with 5 µM CPT for 2 h and further recovery for 1 h. The cells were fixed and subjected to immunostaining and quantification analysis (right). The percentages of cells with more than 10 DDX1 foci were quantified. Scale bars for overall and magnified images are respectively 50 and 10 µm. c HEK293T cells transfected with GFP or GFP-SART3 were treated with CPT (5 µM, 2 h), followed by recovery and subsequent immunoprecipitation and immunoblotting. d U2OS cells stably expressing Flag or Flag-RNase H1 were transfected with siNC or siSART3. Cells were microirradiated and fixed, followed by co-immunofluorescence staining with anti-γH2AX and anti-DDX1 antibodies. Representative images are shown (left). Scale bar, 10 µm. e HEK293T cells expressing various of GFP-SART3 truncations were lysed in the presence of RNase A and Benzonase, followed by immunoprecipitation and immunoblotting. f HEK293T cells transfected with the indicated constructs were lysed for immunoprecipitation, followed by immunoblotting. g HEK293T cells transfected with the indicated constructs were harvested and incubated with biotin-DNA‒RNA hybrids. Pulled-down proteins were analyzed by immunoblotting, and GFP-SART3 levels were quantified by Image J, normalized to input SART3. h, i U2OS cells stably expressing GFP, GFP-SART3 (WT), GFP-SART3-R836W (h) or GFP-SART3-T4 (i) were transfected with siNC or siSART3. Cells were then treated with CPT, followed by co-immunofluorescence staining with anti-DDX1 and anti-γH2AX antibodies. The percentages of cells with more than 10 DDX1 foci were quantified (middle). Representative images are shown (left). Scale bars for overall and magnified images are respectively 50 and 10 µm. The knockdown efficiency of SART3 was determined by Western blotting (right). Error bars represent the mean ± SEM, N = 3 (b, h, i) or 4 (d) independent experiments, and p values were calculated using unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.