Telomere Uncapping and DNA Damage Assays

For telomere uncapping assays, MT-hTer-47A shRNA cell lines were was generated as described in Stohr and Blackburn (2008) (link) and cells were fixed for imaging one week after initial infection. For global DNA damage induction via UV, cells were washed three times in calcium- and magnesium-free Phosphate-Buffered Saline (PBS). The PBS was removed and cells were exposed to 50 J/m-s UV in a hybridization oven. After exposure, cells were washed once and complete medium was added. UV-treated cells were then incubated at 37°C with 5% CO₂ for 6 hr and then fixed for imaging. For neocarzinostatin (NCS; Sigma) treatment, NCS was added to complete medium to a final concentration of 50 pg/ml. Cells were washed in PBS and NCS-supplemented media was added; cells were then incubated at 37°C with 5% CO₂ for 6 hr and fixed for imaging. For methyl methanosulfonate (MMS; Sigma) treatment, MMS was added to complete medium at a final concentration of either 0.01% or 0.05% vol/vol. Cells were washed in PBS and MMS-supplemented media was added; cells were then incubated at 37°C with 5% CO₂ for 2 hr or as indicated before fixation for imaging.