Fig. 6. C-Nap1 overexpression does not prevent cell cycle progression. (A) U2OS cells were co-transfected with two plasmids, one encoding a non-destructible cyclin B2 mutant and the second C-Nap1, GFP or Cdk1-DN, as indicated. At 36 hours post-transfection, cells were fixed and the percentages of cells in interphase (black bars) and mitosis (light gray bars) determined. Transfected cells were detected using the 9E10 mAb for C-Nap1 and anti-HA mAb for Cdk1-DN. Enhanced GFP was detected using the FITC filter. In two independent experiments, more than 200 cells were counted. (B) Integration and expression of GFP-C-Nap1 in TREx modified U2OS cells. Expression of the fusion protein was induced for 24 hours with 1 µg/ml tetracycline. Induced (i) or uninduced (u) cells were lysed directly in sample buffer, and extracts were analyzed by immunoblotting using the indicated antibodies. The positions of molecular weight markers are indicated (in kDa) and the arrowhead marks C-Nap1. (C) Growth rates were determined for stable cell lines in which the expression of GFP-C-Nap1 had not been induced (uninduced), or induced (1 µg/ml tetracycline) at either time 0 (induced) or 24 hours before the initiation of the experiment (pre-induced). Cells were collected at the times indicated and counted.

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