Fig. 2: TUG1 interacts with pRPA32 at R-loop.
a Super-resolution images of the nucleus (dotted line) co-stained with pRPA32 (green) and TUG1 smFISH (red). (i-ix) HeLa cell treated with DMSO (untreated control), or 2 mM HU for 2 h, or 10 μM CPT for 2 h. (x-xv) U2OS cell treated with DMSO or 10 μM CPT for 2 h. (xvi-xxi) U251MG cell treated with DMSO or 10 μM CPT for 2 h. Magnified regions where TUG1 and pRPA32 colocalize in the top panels were shown in the bottom two panels. Three independent experiments were carried out with similar results (Supplementary Table 1) and representative images are shown. Scale bar = 5 μm. b The percentage of TUG1 spots colocalizing with pRPA32 relative to total number of TUG1 spots in each cell. c Left, representative super-resolution images of TUG1 (red) and γ-H2AX (green) in HeLa cell treated with 10 μM CPT for 2 h. Magnified regions in the left panel were shown in middle (i) and right (ii) panels. Scale bar = 5 μm. Right, the percentage of TUG1 spots colocalizing with γ-H2AX relative to the total number of TUG1 spots in each cell. d RIP assay using GFP antibody indicating the interaction between RPA32 or RPA70 and TUG1 after treatment with 10 μM CPT in HeLa cells expressing RPA32-GFP or RPA70-GFP. HPRT and IgG-bound RNA were taken as negative controls. Data are mean ± SD, n = 3. Two-sided t-test. e Left top, super-resolution images of HeLa cell transfected with catalytically inactive RNase H1 (RNH1D210N-GFP, green) co-stained with pRPA32 (white) and TUG1 smFISH (red). Cell was treated with DMSO or 10 μM CPT for 2 h. Left bottom panels are magnified regions. Scale bar = 5 μm. Right, the percentage of TUG1 spots colocalizing with RNH1D210N-GFP relative to the total number of TUG1 spots in each cell. For b, c, and e, median, upper and lower quartile range from 10 independent cells are indicated. Two-sided t-test. Three independent experiments were carried out with similar results (Supplementary Table 1) and a representative image is shown. Source data are provided as a Source Data file.
