Figure 5: WHSC1L1-mediated K721 mono-methylation of nuclear EGFR enhances DNA replication.

(a) 293T cells were transfected with HA-WHSC1L1 and FLAG-EGFR-WT versus HA-WHSC1L1 and FLAG-EGFR-K721A vectors. 24 h after transfection, cells were transferred to collagen-coated immunocytochemistry slides and were exposed to aphidicolin (5 μg/ml) for 24 h to synchronize the cell cycle. At 48 h from transfection, the cell cycle was released by removal of aphidicolin and 3 h post-release, when peak of the S phase is expected, the EdU imaging kit was initiated, while cells were also stained for FLAG expression. Fluorescence intensity was analyzed using the Image J software and corrected total cell fluorescence was calculated as follows: Integrated density – (area of selected cell x mean fluorescence of background). The average EdU fluorescence intensities were compared in 293T cells transfected with FLAG-EGFR-WT (average = 4022 ± 1310, standard error (SE)) versus FLAG-EGFR-K721A (average = 2395 ± 596 (SE)) and results are shown with a histogram (ANOVA test, *p = 0.0358). (b) YD-10B cells were transfected with FLAG-EGFR-WT versus FLAG-EGFR-K721A and at 24 h, they were exposed to aphidicolin (5 μg/ml) for cell cycle synchronization. After 24 h, aphidicolin was released and BrdU exposure was performed at 6 h post-aphidicolin release to capture the S-phase of YD-10B cells. Cells transfected with FLAG-EGFR-K721A showed a decrease in the S-phase percentage of cells compared to cells transfected with FLAG-EGFR-WT. The percentages of cells in each cell cycle phase are also shown in a histogram representative of this experiment. This experiment was duplicated.