Clear 12 well plate

3×10⁵ JURKAT-eDHFR-YFP cells were seeded in a clear 12-well plate (Falcon) in complete media and incubated overnight. The next day, all wells were dosed with 100 nM of 7c, and cells were sampled at 0, 4, 8, 12, and 24 h following incubation (1 well was sampled per time point). Following 24 h incubation, the remaining wells of cells were collected and centrifuged at 200 xg for 5 min (Thermo Scientific Sorvall Legend X1R). Cells were washed 3 times with PBS (Corning) and seeded on a new, clear 12-well plate (Falcon) in fresh complete media. The cells were sampled at 3, 6, 24, 48, and 72 h following the drug washout. All cells were fixed in 4% paraformaldehyde (PFA; Sigma) following sampling, and samples from all time points were analyzed together on a flow cytometer (LSR II, BD).

3×10⁵ HEK293T-eDHFR-YFP cells were seeded in a clear 12-well plate (Falcon) in complete media and incubated overnight. The next day, the cells were incubated with 100 nM 7c for 24 h. Following incubation, media was removed by vacuum, cells were gently washed twice with PBS (Corning), and returned to culture in complete media. Cells were harvested at various time points before and after drug washout and prepared for Western blot analysis.