Calcium-dependent NF- B activity during cell cycle | Sée et al. 663 Figure 1. NF- B dependent transcription is essential for cell cycle progression. (A) Swiss 3T3 fibroblasts were serum starved for 24 h. They were then stimulated with 10% FCS for 18 h in the absence or presence of an NF- B inhibitor (5 M Bay117082). Cell cycle stage was determined by flow cytometry analysis of propidium iodide–stained cells. (B) Swiss 3T3 cells were transfected with the indicated reporter vectors (NF- B luc and 1745 CD1-luc). 24 h after serum starvation, firefly luciferin (Biosynth) was added to the medium (0.5-mM final concentration). 2 h later, serum stimulation was performed and luminescence imaging was carried out using a Hamamatsu 4880-65 liquid nitrogen–cooled CCD camera. Images were acquired using 30-min integration times. (C) RT-PCR analysis was performed using primers specific to CD1 or cyclophilin A (control), with total RNA prepared from cells stimulated with 10% FCS at indicated time points. (D) RT-PCR analysis, with total RNA prepared from nonstimulated starved cells (ct) or cells stimulated 8 h with 10% FCS in the presence or absence of Bay117082. loading the cell-permeant fluorescent dye Fluo-4-AM. Se- rum induced an immediate and significant peak in [Ca2 ]i (Fig. 4 A). The transient increase in [Ca2 ]i started 1 s after serum stimulation and lasted for 30 s. No further oscillations of [Ca2 ]i were observed (over 5 min; unpublished data). We used a calcium chelator (BAPTA-AM) to investigate the effect of ablation of the serum-induced calcium signal on p65-dsRed translocation. 10 M BAPTA-AM was loaded into the cells for 20 min and the cells were then stimulated with 10% serum. Treatment with the calcium chelator led to a drastic inhibition in the level of p65 translocation in com- parison to control cells (Fig. 4 B). The ratio of the nuclear/ cytoplasmic fluorescence intensities reached a maximum of 1.6 in control conditions versus 0.4 in presence of BAPTA. BAPTA can inhibit many calcium-dependent events in cells, leading to nonspecific toxicity after 4 h. To be able to study the long-term role of the serum-induced [Ca2 ]i in- crease, we developed a noninvasive method to abolish the