Fancc^–/– BMMCs enter S phase earlier than WT controls. (A) Percent G0 BMMCs after serum/cytokine starvation. WT and Fancc^–/– BMMCs were quiesced overnight in RPMI 0.1% FCS before analyzing G0 status using Hoeschst 33342 and Pyronin Y. The mean of 5 independent experiments is shown, *P < .01. (B) S-phase entry of BMMCs after serum/cytokine starvation. After an overnight period of quiescence, WT and Fancc^–/– BMMCs were stimulated with 100 ng/mL mSCF in the presence of ³H-thymidine. Cells were harvested at the time points shown and β emission was measured. The mean of 3 experiments is shown, **P < .001. (C) PKH26 staining of BMMCs to verify cell division. WT and Fancc^–/– BMMCs were loaded with PKH26 for 5 minutes, washed, and stimulated with 100 ng/mL SCF for 2 days. BMMCs were analyzed by fluorescence cytometry for PKH26^high (few cell divisions) and PKH26^low (most cell divisions) cells. A representative experiment and the mean of 4 experiments are shown, *P < .05. (D) Retinoblastoma Western blotting. After an overnight period of quiescence, WT and Fancc^–/– BMMCs were stimulated with 100 ng/mL mSCF. Cells were harvested at the time points indicated and evaluated by Western blotting for retinoblastoma (top autoradiograph). Rb hyperphosphorylation (top arrow) can be distinguished from unphosphorylated Rb (bottom arrow) by the retardation of protein migration. The β-actin loading control is shown in the bottom autoradiograph. These data represent 1 of 5 separate experiments with similar results. A Student t distribution and P values were determined using GraphPad Prism 3.0a software.