FIG. 5.

c-Myc expression is tightly regulated in synchronized HCT116 cells. (A) FACS profiles of propidium iodide-stained HCT116 cells that were serum deprived for 48 h and then cultured in the presence of serum-containing medium for the number of hours indicated at the top of each panel. FL2-H is peak emission values of propidium iodide-stained DNA fluorescence. (B) RNAs were isolated from HCT116 cells treated as described for panel A, and cDNA was synthesized using a random primer and avian myeloblastosis virus reverse transcriptase. Real-time quantitative PCR was conducted using primers designed against the third exon of c-Myc or the fourth exon of tubulin as an internal control. The data are presented as relative c-Myc mRNA levels normalized to tubulin. Error bars indicate standard errors of the means. (C) Protein extracts prepared from HCT116 cells synchronized as in panel A were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with anti-c-Myc, anti-β-catenin, anti-TCF4, or antitubulin antibodies in a Western blot.