FIG. 6.

Influences of CSIG levels on replicative senescence. (A) Early passage 2BS (∼28 pdl) cells were transfected with either pIRES-CSIG (CSIG) versus empty vector (vector) (left panel) or pSilencer-CSIG (CSIG siRNA) versus vector expressing Ctrl siRNA (siRNA Ctrl) (right panel) and then selected by G418 for 3 weeks. Expression of CSIG was monitored by Western blot analysis; β-actin served as a loading Ctrl. Relative CSIG abundances were assessed by densitometry and are expressed as increases relative to CSIG levels in cells transfected with the empty vector. (B) Effect of overexpression (left panel) or silencing (right panel) of CSIG on cell growth. Transfected cells were seeded 2 × 10³ per well in 96-well plates, and cell number was assessed by MTT (methyl thiazolyldiphenyl-tetrazolium bromide) method at the times indicated. Values represent the means ± SEMs of the results for three independent experiments. (C) Transfected cells were subjected to FACS analysis to evaluate the effect of ectopic intervention to either overexpress (left panel) or knock down (right panel) CSIG on cell cycle distribution. Bars represent the means ± SEMs of the results for three independent experiments. (D) Influence of CSIG levels on SA-β-gal activity. Transfected 2BS cells that either elevate (left panel) or reduce (right panel) CSIG levels were stained to assess SA-β-gal activity. Data are representative of the results for three independent experiments.