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

Figure 2.

Figure 2.

ATAD5 depletion increases PCNA-RNAPII PLA signals and reduces transcription rate. (A, B) HeLa cells were transfected with ATAD5 siRNA and, after 24 h, transfected again with GFP cDNA or GFP-tagged RNaseH1 cDNA. 24 h later, cells were fixed and subjected to a proximity ligation assay (PLA) between PCNA and RNA polymerase II (RNAPII). (A) Representative images of PLA. (B) Number of PLA nuclear foci was counted and plotted. (C, D, G, H) HeLa cells were transfected with ATAD5 siRNA for 48 h before harvesting or fixation. To inhibit transcription, cells were exposed to 100 μM cordycepin (CORD) for 2 h before fixation. (E, F) HeLa-TetOn-RNaseH1-GFP cells were transfected with ATAD5 siRNA, treated with doxycycline (DOX) to express RNaseH1 for 48 h before fixation. (C–F) Cells were treated with 1 mM 5-ethynyluridine (5-EU) for 10 min before fixation and subjected to EU-click reactions. (C, E) Representative images of EU staining. (G, H) Fixed cells were subjected to S9.6 immunostaining. (G) Representative images. (B, D, F, H) 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 and ns, not significant.