FKBP12−/− fibroblasts are arrested in G1
phase of the cell cycle. (A) Cell growth. For
FKBP12+/+ or FKBP12−/− cells, 5 ×
104 cells were plated in six-well dishes and counted in
24-h intervals by hematocytometry. Trypan blue dye was used to count
viable cells. Data points are the mean ± SEM from three
experiments. (B) Cyclin D1 staining.
(Left) Nuclear localization of cyclin D1 was observed in
more than 80% of FKBP12−/− cells. (Right)
The bar graph presents the mean ± SEM from three experiments,
with at least 200 cells counted in 10 fields for each experiment
(*, P < 0.001). (C) Cell
cycle progression in FKBP12−/− cells stimulated with bFGF
(25 ng/ml) was monitored by using cyclin D1 (G1 phase)
and PCNA (S phase) as markers. In FKBP12+/+ cells, a 24-h
bFGF treatment stimulates expression of cyclin D1 4-fold (bar b is
different from bar a, P < 0.001) and expression of
PCNA 3.5-fold (bar d is different from bar c, P <
0.001), indicating an increase in cells in S phase. In
FKBP12−/− cells, cyclin D1 is stimulated 5-fold (bar d
different from bar c, P < 0.001) with only a
modest increase in PCNA, indicating G1-phase blockade and
failure to progress to S phase. Values are the mean ± SEM of five
determinations.