668 The Journal of Cell Biology | Volume 166, Number 5, 2004 Figure 6. A photoactivated induced [Ca2 ]i peak is not sufficient to promote p65 translocation. (A) Starved cells were loaded with both 0.6 M NP-EGTA and 0.6 M Fluo-4 for 20 min at 37 C. For uncaging of NP-EGTA, cells were illuminated for 10 s with a Micro-point photo- activation 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) Subse- quently, p65 localization was assessed every 2 min for 90 min as already described. The control (ct) is nonilluminated cells stimulated with serum. 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. occurred in the first few hours after serum stimulation (i.e., at the G0–G1 transition). The small difference in timing ob- served in the dynamics of the NF- B–luc and CD1-luc ac- tivity (peak of activity at 7 and 3 h, respectively) was presum- ably due to the contribution of other transcription factors to the activity of the CD1 promoter. The timing of NF- B ac- tivity agreed with a previous report of DNA-binding kinetics (Baldwin et al., 1991). Therefore, these data were consistent with the hypothesis that NF- B plays an important role in the early G0/G1 stage of the cell cycle. Further evidence for the role of NF- B in the regulation of CD1 transcription in response to serum was obtained from deletion and mutation of the NF- B sites in the CD1 promoter, which was found to reduce serum activation, identifying the key role for these elements in this process. Further evidence was obtained from overexpression of a truncated IKB or transdominant-nega- tive IKK and proteins (Fig. 2 B). IKK has recently been shown to activate transcription of CD1 via direct activation of Tcf in mouse embryo fibroblasts upon serum stimulation (Albanese et al., 2003). In the absence of IKK (IKK / Figure 7. I B degradation is both [Ca2 ]i and p42/p44MAPK activity dependent. (A) I B degradation, p42/p44MAPK phosphorylation, and Akt phosphorylation were determined by Western blotting after 10 min of serum stimulation, using antibodies directed against I B (1:1,000), phospho-p42/p44 MAPK (Thr185/202/Tyr185/204; 1:5,000), and phospho-Akt (Ser 473; 1:5,000) and actin (1:10,000). Swiss 3T3 fibroblasts were serum starved for 24 h before serum stimulation, and the pharmacological inhibitors were added, where indicated, 20 min before the serum stimulation at the following concentrations: PD98059, 50 M; BAPTA-AM, 10 M; wortmannin, 100 nM. (B) The kinetics of p42/p44MAPK and Akt activation were determined by Western blotting using anti-phospho-p42/p44 MAPK and anti-phos- pho-Akt antibodies. 24-h serum-starved 3T3 cells were stimulated by 10% FCS for indicated time points before cell lysis. cells) the CD1 induction was delayed by about 3 h, perhaps suggesting an upstream reinforcement mechanism by IKK to the control of the CD1 promoter. We used siRNA abla- tion of p65 to attempt to show the importance of this factor for CD1 transcription. However, we did not see any signifi- cant affect on CD1 transcription (Fig. S3, available at http:// www.jcb.org/cgi/content/full/jcb.200402136/DC1). Despite good knock-down of this stable protein at the mRNA and protein levels, some protein remained, and therefore we can- not exclude the possibility that this was sufficient for func- tion. It has also been shown that other NF- B family mem- bers can activate the CD1 promoter (Guttridge et al., 1999; Westerheide et al., 2001; Rocha et al., 2003; Romieu-Mourez et al., 2003), and indeed the evidence for a critical role for p65 is therefore lacking. In the present work, we have also shown the essential role of MAPK activity in cell cycle progression (Fig. 8 B) because inhibition of MAPK activity with PD 98059 blocked entry into the S-phase of the cell cycle. We observed a persistent p42/p44MAPK activity upon serum stimulation (at least 3 h; Fig. 8 B), which appeared to be causally related to CD1 tran-