Figure 6. LIN54 CHC mutants lead to nuclear aberrations and impede cell cycle progression. (A) Examples of nuclear abnormalities in GC-1 cells expressing FLAG-LIN54C525Y. At 36 h after infection, cells were fixed and stained with DAPI and FITC-phalloidin that binds specifically to F-actin. The arrows indicate cells with abnormal nuclear morphology. (B) GC-1 cells expressing EGFP (filled diamond), FLAG-LIN54 (open square), FLAG-LIN54(ΔCXC1/2) (open diamond), FLAG-LIN54C525Y (open circle) or FLAG-LIN54C611Y (filled triangle) were seeded at 1 x 105 cells per dish in triplicate and counted at indicated time points. The experiment was performed three times. One representative experiment is shown. Error bars represent standard deviation. (C) GC-1 cells were infected with recombinant adenoviruses expressing the indicated constructs. At 24 or 36 h after infection, the cells were fixed and stained with PI. The cell cycle profiles were determined by flow cytometry. (D) GC-1 cells were infected with recombinant adenoviruses expressing EGFP (black bars), FLAG-LIN54 (white bars), FLAG-LIN54(ΔCXC1/2) (dark gray bars), or FLAG-LIN54C525Y (light gray bars). At 24 h after infection, cyclin A2, cyclin B1 and Cdc2 expression were analyzed by quantitative RT-PCR. Relative expression was normalized to Gapdh. Error bars represent standard deviation.