FIG. 2.

Characterization of U2OS cells conditionally coexpressing c-Myc and pRbΔcdk. (A) Immunoblotting with MAbs to pRb and c-Myc. Expression of the ectopic proteins is tightly repressed in a representative U2OS-RbΔcdk/Myc clone (clone A4C7) exposed to TET (day 0), as opposed to the high levels observed during the induction time. endo pRb, endogenous pRb. Endogenous c-Myc migrates as a faint band just below the ectopic one. (B) Endogenous E2F activity examined by in vivo photon emission imaging in cells injected with 6xE2F-Luc reporter before (day 0) or after (day 4) coinduction of transgenes. The average emission per cell during the acquisition period (10 min) was (24 ± 15) × 10³ and (0.5 ± 0.3) × 10³ light counts at days 0 and 4, respectively. The background value was (0.4 ± 0.2) × 10³ light counts. (C) Myc stimulates S-phase entry in cells deprived of E2F activity. Representative flow-cytometric DNA histograms of propidium iodide-stained U2OS-RbΔcdk/Myc cells grown with (day 0) or without TET for the indicated times. The cell cycle distribution at each time point is indicated above the histograms. As in Fig. 1C, the numbers in parentheses represent the corresponding distribution of U2OS-RbΔcdk cells at the same time points. The asterisk is as defined in the legend to Fig. 1C. Note the presence of pre-G₁ (apoptotic) cells and endoreplication after transgene induction in U2OS-RbΔcdk/Myc cells. The time course was reproduced at least three times in this clone and in other independent clones.