RNA Synthesis Assay

Cells were grown in a 4-well chamber slide and exposed to THS for 24 h. RNA synthesis levels were determined by directly adding 5-ethynyl uridine (EU; 1 mM final concentration) to the culture medium and incubation for 2 h, with incorporation of EU visualized by Click IT conjugation of Alexa Fluor 488 according to the manufacturer’s protocol described earlier (Invitrogen Cat# c10329 and Nakazawa et al., 2010). Images were obtained using Zeiss LSM 710 confocal microscope as described above. Fluorescence-signal intensities were quantified using the ImageJ software (NIH). For each experiment, >100 cells were used for analysis.