FIG. 1.

Cyclin G2 mRNA levels are maximal in G₀. (A to C) NIH 3T3 cells were arrested by confluence and then released by low-density replating in medium with serum for different time periods. (A) Representative cell cycle profiles of cells 0, 18, and 23 h after release, showing the percentage of cells in G₀/G₁, S, and G₂/M. (B) Western blot analysis of the NIH 3T3 cells entering the cell cycle synchronously, using anti-cyclin D3 (cycD3), anti-p130, anti-phospho Ser 473-PKB (pPKB), or anti-PKB antibody. (C) Northern blot analysis of cyclin G2 expression. Total RNA was extracted from NIH 3T3 cells (same as in panels A and B), resolved in agarose gels (10 μg), transferred, and hybridized with a probe for cyclin G2 or 28S rRNA. The percentage of cells in the S and G₂/M phases is indicated. (D) Representative FKHRL1 (FoxO3a) immunofluorescence staining of NIH 3T3 cells deprived of serum for 18 h (−serum) or deprived of serum for 18 h and then incubated with serum for 1 h (+serum). (E and F) NIH 3T3 cells deprived of serum for 18 h (time 0), deprived of serum for 18 h and then incubated with serum for different times (indicated in minutes), or arrested in the G₂ or M phase. (E) Western blot analysis using the indicated antibodies. (F) Northern blot analysis of cyclin G2 expression. Total RNA was extracted from aliquots of the cells used in panel E. The percentage of cells in the S and G₂/M phases is indicated. The figure illustrates a representative experiment of at least three with similar results.