rRNA, DNA, and nascent peptide synthesis in vitro

FUrd assay was performed as previously described^28,29. Cells were pulsed with 2 mM FUrd for 8–10 min following 48 h of treatment (TGFβ or hypoxia), under normal culturing conditions. Following the pulse, cells were rinsed with PBS and fixed with paraformaldehyde. In addition, cells were pulsed with 20 μM EdU for 45 min according to manufacturer instructions under normal culturing conditions, following the pulse cells were rinsed with PBS and fixed with paraformaldehyde. Click-it assay was performed as specified by manufacturer instructions. Briefly, cells grown in methionine-free media were pulsed with the AHA amino acid analog for 30 min. Cells were fixed with paraformaldehyde for 15 min, permeabilized with 0.3% triton-PBS for 15 min and then the incorporated analogs were labeled via Click-it chemistry using the Alexa Fluor 488 azide. Cells were then washed with PBS, stained with DAPI and mounted. Each experiment was performed on 3 biological replicates. Significance was assessed with two-tailed Student’s t-test.