Click it plus edu cell proliferation kit for imaging alexa fluor 555 dye

Click-iT Plus EdU Cell Proliferation Kit for Imaging (Alexa Fluor^™ 555 dye) was obtained from Thermo Fisher Scientific and reagents prepared according to manufacturer’s instructions. HEKn cells were plated in 4-well chamber slides at 1.25×10⁴ cells per well or 2.5×10⁴ cells per well (siRNA experiments only) and treated as described in figure legends. Ten μM EdU was added 1 h prior to UV exposure and cells were fixed 4 h post UV for a total EdU treatment of 5 h. Cells not exposed to UV were also treated with EdU for 5 h. EdU detection was performed according to manufacturer’s instructions. Briefly, cells were fixed in 3.7% paraformaldehyde for 15 min, washed 2 times with 3% BSA in 1xPBS, then permeabilized with 0.5% Triton^® X-100 in 1xPBS for 20 min. Cells were washed 2 times with 3% BSA in 1xPBS, then incubated with Click-iT Plus reaction cocktail for 20 min. Cells were washed once with 3% BSA in 1xPBS, then 2 times with 1xPBS prior to a 30 min incubation with Hoechst^® 33342 in 1xPBS. Cells were washed 3 times with 1xPBS and mounted with Invitrogen Prolong Glass Antifade Mountant. All incubations were performed on a rotator at room temperature. Slides were allowed to cure overnight at room temperature, then sealed with nail polish and stored at 4°C. Imaging was performed as described in 2.5.