Fig. 6. Impaired proliferation and deregulated expression of cell cycle regulators in junB^–/– Ubi-junB cultures. (A) Growth rate of bone marrow-derived control (filled symbols) and junB^–/– Ubi-junB (open symbols) stromal cells. 1x10⁴ cells were plated in 96-well plates and total number of cells was measured at indicated time points. Values are means ± s.e.m. of three independent experiments done in duplicates. (B) Reduced number of BrdU-positive cells in junB^–/– Ubi-junB stromal cell cultures. Cells were cultured in the presence of 60 µM BrdU for 2 hours and the BrdU-positive cells were analysed by immunohistochemistry with an anti-BrdU antibody. Bars represent mean ± s.e.m. of two independent experiments done in triplicates. (C) Expression of cell cycle regulators (Cyclin A, Cyclin D1, p16^INK4 and p21) was measured by RT-PCR analysis with cDNA prepared from asynchronously growing control (lane 1) and mutant (lane 2) stromal cells. RT-PCR analysis for JunB demonstrates lack of JunB expression in bone marrow-derived stromal cell cultures and ß-Tubulin served as control for quantity and quality of used cDNA samples.