Calcium-dependent NF- B activity during cell cycle | Sée et al. 667 Figure 5. The serum-induced [Ca2 ]i peak is essential for NF- B–dependent signaling. (A) Starved cells were loaded with both 0.6 M Diazo-2 and 0.6 M Fluo-4 for 20 min at 37 C. For uncaging Diazo-2, cells were illuminated for 10 s with a Micro-point photoactivation system (Photonic Instruments). Intracellular calcium content was monitored every second by confocal microscopy (488-nm excitation, 505–550-nm emission) of Fluo-4 fluorescence. (B–D) Swiss 3T3 cells were plated on a marked dish and transfected with 0.5 g p65-dsRed and 1.5 g of the reporter NF- B–luc. After 24 h of serum starvation, the cells were loaded with both Fluo-4 and Diazo-2 as previously described. After one wash, the cells were stimulated with 10% FCS. 1 s later the cells were either illuminated for 10 s (open squares) or untreated (ct, filled squares). (B) Confocal microscopy was used as previously described to measure calcium content every second for 2 min. (C) p65 localization was assessed every 2 min for 90 min as already described. (D) An intensified CCD camera (VIM; Hamamatsu Corporation) was used to assess the luminescence from fields of individual control or photoactivated cells as previously described. Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells. The horizontal line in A and B marks the period of photoactivation. cycle kinetics were significantly affected by stable transfec- tion of an expression vector for DA-I B . We confirmed the previously identified role of NF- B in regulation of CD1 transcription (Fig. 1; Guttridge et al., 1999; Hinz et al., 1999). The use of luminescence imaging with a highly sensitive CCD camera allowed us to follow CD1 and consensus NF- B promoter activities throughout the cell cycle. We observed two peaks of CD1 promoter ac- tivity within a 20-h interval, which appeared to correspond to the completion of one cell cycle. The NF- B vector only showed one luminescence peak after serum stimulation, ap- parently indicating that the increase in NF- B activity only