FIGURE 7:

Tetraploid HFFs express quiescence and senescence markers. (A) Primary cilia, markers of cell quiescence, become abundant in HFF cells that have been exposed to 10 μM DCB for 24 h (DCB treated), indicating quiescence of both mononucleate and binucleate cells. By 72 h of release from DCB, the transiently arrested mononucleate population returns to nontreated (NT) levels, whereas the binucleate population remains ciliated. Serum-starved G0 HFFs serve as a positive control. Right, immunofluorescence of primary cilia in binucleate HFF cells, visualized with antibody to acetylated α-tubulin (green; Pugacheva et al., 2007). Counterstains were total α-tubulin (red) and DNA (DAPI, blue). Scale bar, 20 μm. (B) Tetraploid HFFs are positive for SA-β-galactosidase by 3 d after release from DCB. HFFs were treated with 10 μM DCB for 24 h and released. Cells were harvested and assayed for SA-β-gal activity at time points indicated. The percentage SA-β-gal–positive binucleate cells was quantitated in three independent experiments. Results are expressed as mean ± SD. Right, images of binucleate cells stained for SA-β-gal activity at 3 d of release compared with untreated controls. Scale bar, 40 μm.