Calcium-dependent NF- B activity during cell cycle | Sée et al. 665 Figure 3. Serum stimulation promotes p65 translocation into the nucleus as well as I B degradation. (A) p65-dsRed (red staining) and I B -EGFP (green staining) were cotransfected into Swiss 3T3 fibroblasts. 24 h after serum starvation, the cells were stimulated with 10% FCS. Confocal images were collected every 2 min from transfected living cells cultured in a humidified CO2 incubator (5% CO2, 37 C) (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200402136/DC1). (B) Mean fluorescence intensities were measured for each time point in both the nucleus and cytoplasm for p65-dsRed, and the results are shown as a ratio. The I B -EGFP mean fluorescence intensities were measured at each time point in the cytoplasm alone and the results presented as the relative intensity to the starting fluorescence levels. Experiments were performed at least four times, with four fields. In each field there were typically 3–4 transfected cells. (C) Endogenous p65 levels in the nucleus were assessed after 10% FCS stimulation for the indicated time by Western blotting nuclear extracts using an anti-p65 antibody (1:1,000). Endogenous I B was assessed in whole-cell extracts using an anti-I B antibody (1:1,000). caged calcium donor. Photoactivation of NP-EGTA in- duced a 22% transient increase above the resting level in [Ca2 ]i (Fig. 6 A). This increase was smaller, but of the same magnitude to that obtained with serum stimulation (22% with illumination compared to 60% above the resting level with serum). The increase in [Ca2 ]i after illumination lasted for a similar duration to that obtained with serum stimulation ( 20 s). Despite the similar dynamics in [Ca2 ]i levels (compared to serum stimulation), the illumination of NP-EGTA was unable to induce any detectable p65 translo- cation (Fig. 6 B). Together, these data (Fig. 5 and Fig. 6) suggest that p65 translocation and downstream regulation of gene transcription is dependent on a transient increase of [Ca2 ]i. However, that increase alone, without the presence of other serum-dependent signals, was not sufficient to acti- vate p65. Therefore, we next set out to investigate which other factor(s) upstream of I B degradation were activated by growth factors and were dependent on calcium signaling. Serum-induced NF- B activity is dependent on calcium-sensitive p42/p44MAPK activity Candidate signaling enzymes that might be involved in the serum-induced NF- B activity were p42/p44MAPK and Akt, which are known to be activated during cell proliferation (Pages et al., 1993; Davies et al., 1999), and have been re- ported to regulate the I B/NF- B signaling pathway (Ro- mashkova and Makarov, 1999; Madrid et al., 2001). Other candidates were investigated including Ca-calmodulin ki- nases (CaMKs), calcineurin, and calmodulin. Inhibition of these either had no significant effect (cyclosporin A and