Fig. 3: Tpr depletion leads to DNA–RNA hybrid accumulation.

a Tpr-depleted (siTPR) and control (siCTRL) cells. Treatments: nontreated (Mock); 2 mM hydroxyurea 4 h (HU); 50 μM cordycepin 3 h followed by 2 mM HU 4 h (HU + cord). RPA total, loading control. Data are representative of three independent experiments. Source data are provided in the Source Data file. b Quantification of γH2AX-foci in siTPR (red) and siCTRL (blue) HeLa cells. Data represent mean ± SEM from independent experiments (n = 3). Statistical analysis: two-tailed unpaired Student’ t test. c Quantification of 53BP1 foci in siTPR and siCTRL HeLa cells. Other details as in b. d DNA breaks in Tpr-depleted cells are transcription dependent. Alkaline comet assay in mock or cordycepin-treated (+Cord) siTPR (red) or siCTRL (blue) HeLa cells. Representative images (left) and mean ± SEM of median comet tail moment (right) from independent experiments (n = 3). Statistical analysis: two-tailed unpaired Student’ t test. e RNaseH1 expression rescues DNA breaks in Tpr-depleted HeLa cells. Alkaline comet assay in siTPR (red) and siCTRL (blue) cells expressing RNaseH1 from plasmid pEGFP-M27-H1 (+RNH1) or transfected with plasmid pEGFP-N2 (−RNH1). Other details as in d. f DNA–RNA hybrid immunoprecipitation with the S9.6 antibody in siTPR (red) and siCTRL (blue) HeLa cells at the APOE, RPL13A, EGR1, and BTBD19 genes. Where indicated samples were treated with RNase H (RNH) prior to immunoprecipitation. S9.6 signal values were normalized to the control cells value of each locus in each experiment. Location of the amplicon relative to each gene is indicated. Mean ± SEM of signal values of R-loop detection obtained from independent experiments are shown (n = 5, except for siTPR +RNH of RPL13A, siCTRL +RNH, and both siTPR of EGR1, siTPR −RNH of BTBD19 for which n = 4, and siTPR +RNH of BTBD19 for which n = 3). Statistical analyses with a two-tailed unpaired Student’ t test. g siTPR pool deconvolution analysis by S9.6 immunofluorescence in HeLa cells. Immunofluorescence with S9.6 and anti-nucleolin antibodies in siCTRL (blue), siTPR pool (siTPR pool; red), or each individual siRNAs of the siTPR pool (siTPR #1, siTPR #2, siTPR #3, siTPR #4; red) transfected cells. Representative images from three independent experiments (left) and quantification of S9.6 signal intensity per nucleus after the subtraction of the nucleolar signal (right). Scale bar, 7.5 µm. Median value and number of cells (n) examined over three independent experiments are indicated. Statistical analysis: two-tailed unpaired Mann–Whitney U test; ****p < 0.0001.