Cell cycle analysis by BrdU incorporation

The cell proliferation analysis was performed as previously described (Hong et al., 2021). WASH^WT and WASH^KO 3T3 cells at about 80% confluence were treated with 30 μM BrdU for 30 min at 37°C. Then cells were fixed, acid-treated, and detected using FITC-conjugated anti-BrdU antibody. The DNA was stained with 20 μg/mL of propidium iodide (PI). All samples were analyzed using a FACS Calibur flow cytometer (BD Biosciences) and FlowJo software.