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. 2020 Jun 15;48(13):7218–7238. doi: 10.1093/nar/gkaa501

Figure 6.

Figure 6.

Depletion of ATAD5/UAF1-interacting RNA helicases increases R-loop formation under replication stress. (A) HeLa-TetOn-RNaseH1-GFP cells were transfected as indicated and subjected to S9.6 immunostaining. (B, C, E, F) U2OS-TetOn-ATAD5 cell lines were transfected with ATAD5 or UAF1 siRNAs and treated with doxycycline to express wild type or mutant ATAD5. After 48 h, cells were fixed for S9.6 immunostaining. (D) HeLa-FUCCI cells were transfected with a combination of ATAD5 siRNA and ATAD5 cDNA expression vector for 48 h, sorted into G1 and S/G2/M phases using a cell sorter, and immunostained with the S9.6 antibody. (G, H) HeLa cells were transfected with indicated siRNAs before fixation for S9.6 immunostaining. (C–E, G, H) Aphidicolin (APH) was added to cells at a concentration of 0.4 μM for 1 h before fixation. (A–H) The intensity of S9.6 staining was quantified and plotted. Three independent experiments were performed and one representative result was displayed. Red bar indicates mean value. Statistical analysis: two-tailed Student's t-test; **** P < 0.001, *** P < 0.005, **, P < 0.01, * P < 0.05 and ns, not significant.