Nunc 4 well chamber slides

The 293T cells were plated into Nunc 4-well chamber slides (ThermoFisher 154453) and transfected with indicated plasmids and siRNA. After 48 h transfection, cells were fixed in 4% paraformaldehyde at RT for 10 min, followed by 0.1% Triton X-100 permeabilization at RT for 10 min. Cells were then incubated with FLAG antibody (1:100, Sigma, F1804) overnight, then stained with Alexa 488-conjugated secondary antibody (ThermoFisher). Samples were then subjected to DAPI nuclear counterstain (Vector Labs), and imaged by fluorescent microscope.

CaSki cells stably transduced with circE7 shRNA1/2 were induced by 1 μg/mL doxycycline for at least three days. Control cells (no dox) and induced cells were then plated into Nunc 4-well chamber slides (ThermoFisher 154453) in triplicate. Cells were labeled with 10 μM BrdU for 1.5 h. Cells were then fixed by 4% paraformaldehyde at RT for 10 min, followed by 0.1% Triton X-100 permeabilization and 1.5 N HCl DNA hydrolysis. Cells were then probed by O/N incubation with BrdU antibody (1:400, BD BioScience) and stained with Alexa 488-conjugated secondary antibody (ThermoFisher). Samples were then subjected to DAPI nuclear counterstain (Vector Labs). BrdU positive cells were then quantified and data were presented as percentage BrdU positive cells.