Fig 8.

Effect of NEK4 suppression on DNA damage-mediated cell cycle arrest. (A) Colony formation assays to examine the sensitivity of BJ shGFP no. 1 or BJ shN4 no. 1 and no. 2 fibroblasts to treatment with etoposide (top), mitomycin C (middle), or gamma irradiation (bottom). Survival is expressed as the percentage of colonies formed following treatment compared to mock-treated cells. The error bars indicate the standard deviations of triplicate experiments. *, P < 0.015 compared to shGFP no. 1 control. (B) BrdU incorporation assays to assess cell cycle arrest of cells mentioned in part A in response to treatment with etoposide (top), mitomycin C (middle), or gamma irradiation (bottom). The bars indicate the percentages of BrdU-positive cells following treatment with the indicated drug or irradiation, normalized to the percentage of BrdU-positive mock-treated cells. The error bars represent the standard deviations of triplicate experiments. *, P < 0.03; **, P < 0.004; †, P < 0.016; ‡, P < 0.0003 compared to the shGFP no. 1 control. (C) Reexpression of Nek4 restores etoposide-induced cell cycle arrest. A BrdU incorporation assay was performed as shown in panel B using BJ shGFP no. 1 or BJ shN4 no. 1 cells stably transfected with empty vector or Flag-tagged Nek4. Below the bar graph is an immunoblot confirming overexpression of Flag-tagged Nek4 in the appropriate cell lines; actin is shown as a loading control. *, P < 0.024 compared to shN4 no. 1 cells expressing the empty vector.