670 The Journal of Cell Biology | Volume 166, Number 5, 2004 gate the precise role of the calcium increase in response to se- rum stimulation, we initially used calcium chelation by BAPTA (Fig. 4 B). Subsequently, the use of flash photolysis for the transient release of the calcium chelator Diazo-2 al- lowed us to inhibit the serum-induced calcium increase with- out inhibiting other calcium-dependent events in the cell. Control experiments showed that the uncaging approach was specific and noninvasive. First, illumination was not toxic (cell survival was observed in three independent fields of 20 illuminated cells, 20 h after illumination; unpublished data). Second, the inhibitory effect on NF- B and CD1 pro- moter activation was dependent on both illumination and on Diazo-2 loading into the cells. Third, the illumination was only effective when it was performed within a few seconds af- ter serum addition to the cells. Uncaging of Diazo-2 before or 1 min after serum stimulation had no inhibitory effect (Fig. S1). Therefore, these control experiments showed that only the precise experimental conditions that led to ablation of the initial calcium peak were able to inhibit subsequent NF- B activation and CD1 transcriptional activity. We investigated whether a calcium-dependent protein might activate NF- B in response to serum. The role of cal- modulin, CaMKs, and phosphatase (calcineurin) on IKK/ I B/NF- B activity has been widely reported in different cell models (Antonsson et al., 2003; Lilienbaum and Israel, 2003), and they were therefore candidate enzymes for trans- ducing the serum-induced calcium signal. CaMKs and cal- cineurin have also been reported to exert an important role in cell cycle progression (for review see Means, 1994; San- tella, 1998). Because CaMKIV is not expressed in Swiss 3T3 fibroblasts (unpublished data), we tested the role of CaMKII by inhibiting its activity with the well-characterized pharma- cological inhibitor KN93. This inhibitor had no significant effect on serum-induced NF- B–luc transcription (Fig. S2). We also saw no significant effect with the calcineurin inhibi- tor cyclosporin A on serum-induced NF- B–luc transcrip- tion in Swiss 3T3 fibroblasts (Fig. S2). It is notable that most of the previous papers on the relationship between these calcium effector proteins and cell cycle progression re- port a determinant role of calcium in later stages of the cell cycle, such as the G1/S transition (Takuwa et al., 1995) or the G2/M transition (Patel et al., 1999), rather than at the G0–G1 transition. Therefore, we suggest that in early stages of the cell cycle, CaMKII or calcineurin may not be the main effectors for the regulation of CD1 transcription. We have demonstrated for the first time the essential role of calcium in the regulation of CD1 transcription and for com- mitment to cell cycle progression. We found that the early ac- tivated mitogenic p42/p44MAPK is calcium sensitive (Fig. 7 A), and that in absence of the calcium signal there was no activa- tion of the NF- B pathway (IKK phosphorylation, I B phosphorylation, and degradation) or subsequent NF- B– dependent gene transcription. A calcium increase induced by uncaging of caged calcium donor was unable to promote p65 translocation (Fig. 6) and downstream gene transcription (unpublished data). One possible explanation for this would be that the induced calcium increase had a different subcellu- lar localization or dynamics compared to that induced by se- rum. However, we felt that it was more likely that the coacti- vation of another upstream pathway by growth factors is needed for NF- B activation. Present data suggest that the MAPK cascade is therefore a good candidate for this role. In conclusion, this study of the kinetics of signaling and transcription in living cells allowed us to understand the criti- cal role of an early transient event (the serum-induced calcium increase) and its later consequences for the fate of the cells. We used state-of-the-art multiparameter noninvasive imaging techniques to follow different biological processes occurring in different time domains from seconds to minutes and hours in the same cells. We propose that the G0–G1 transition is de- pendent on both mitogenic kinase activity and on calcium sig- naling. NF- B activation was also shown to have a critical role in the transduction of these mitogenic signals to the nucleus. Materials and methods Reagents and antibodies Tissue culture medium was from GIBCO BRL; FCS from Harlan Seralab; pharmacological inhibitors Bay117082, PD98059, wortmannin, and BAPTA-AM from Calbiochem; propidium iodide and ribonuclease A from Sigma-Aldrich; and Fluo-4, NP-EGTA, and Diazo-2 from Molecular Probes, Inc. Antibodies to p65, I B , and phospho-Akt (ser 473) and phospho-p42/ p44MAPK (Thr185/202/Tyr185/204) were from Cell Signaling Technology, Inc.; anti-actin from Oncogene Research Products; HRP anti–rabbit from Cell Signaling Technology, Inc.; and anti–mouse from Amersham Biosciences. Cell culture Swiss albino 3T3 cells were grown in DME supplemented with 10% FCS (vol/vol) and 2 mM L-glutamate at 37 C and 5% CO2. Cells (between pas- sages 6 to 15) were plated at 105 cells/ml and were serum starved (0.1% FCS [vol/vol]) 1 d after plating. 10% (vol/vol) serum stimulation was per- formed 24 h after starvation. Gene transfer Plasmids. The pNF- B-luc construct (Stratagene) contains five repeats of the NF- B consensus sequence upstream of the luciferase reporter. The CD1-luc reporter plasmids were a gift from Dr. R. Pestell (Albert Einstein College of Medicine, Bronx, NY; Albanese et al., 1995). p65-ds-Red and I B-EGFP were described previously (Nelson et al., 2002a). The truncated I B (missing the 40 NH2-terminal amino acids) was cloned into the pIRES2-EFGP vector (CLONTECH Laboratories, Inc.) and was from Dr. E Costello (University of Liverpool, Liverpool, UK). Plasmids expressing the transdominant-negative IKK and were a gift from Dr. N. Inohara (Uni- versity of Michigan Medical School, Ann Arbor, MI; Tang et al., 2003). Transfection. Cells were transfected using FuGENE™-6 (Roche) accord- ing the manufacturer’s instructions. Microplate luminescence assay The cells were plated in 24-well plates and transfected on the same day in presence of serum. After 24 h of serum starvation, the cells were treated for 6 h as described in the figure legends. Cell lysis and luminescence mea- surements were performed as described previously (Nelson et al., 2002b), using a plate reader (LUMIstar; BMG Lab Technologies). The experiments were performed in triplicate. Live-cell imaging of the luminescent reporter gene The cells were plated onto 35-mm glass coverslip culture dishes (Iwaki), transfected with the reporter gene, and subsequently serum starved for 24 h. The cells were then incubated on the microscope stage for 2 h at 37 C, 5% CO2, in the presence of 1 mM luciferin (Biosynth AG) and were stimulated with 10% serum. Imaging was carried out using a microscope (Axiovert 100; Carl Zeiss MicroImaging, Inc.) with a Fluar 20 /0.75 objective. The photons emitted were collected using a CCD camera (Orca II; Hamamatsu Corporation) cooled to 50 C. A series of images was ac- quired using a 30-min integration time over a period of 24 h. AQM2000 software (Kinetic Imaging) was used for image acquisition and analysis. For background correction of luminescence images, Lucida version 6 software (Kinetic Imaging) was used to determine the darkest pixel from a compari- son of two running consecutive images. Subsequently, a control back-