EU incorporation and nascent RNA labeling

To label RNA, a 1h of EU incorporation at 1 mM concentration was performed in HeLa cells, using the Click-iT RNA Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific, #{"type":"entrez-nucleotide","attrs":{"text":"C10329","term_id":"1535400","term_text":"C10329"}}C10329). Cells were plated in chambered coverglass (μSlide Ibidi, #80827) at a concentration of 30.000 cells/cm² in growth medium for 24 h. The following day growth medium was replaced by resting medium and incubated for other 24 h. The third day, cells were cultured in resting medium and treated with ActD at multiple time points or with DMSO for 5h as mock control. During the last hour of treatment, medium was replaced with new treatment medium supplemented with EU working solution.

Cells were fixed with PFA 4% (Alfa Aesar, #43368) for 10 min at room temperature and then rinsed with PBS three times for 5 min each. Cells were permeabilized with 0.3% Triton X-100 in PBS for 15 min and rinsed with PBS three times for 5 min each. Click-iT reaction was performed following manufacturer’s instructions by incubating cells with 1X Click-iT reaction cocktail freshly prepared with Alexa Fluor 488 azide for 30 min at room temperature, protected from light. Cells were washed once with Click-iT reaction rinse buffer and then with PBS. For DNA staining, cells were incubated with Hoechst 33342 at a 1:1000 dilution, for 15 min at room temperature. Cells were washed twice for 5 min each with PBS buffer and then imaged by confocal microscopy (see confocal imaging section).