ground image (processed in the same way) was subtracted from each re- sulting time point. This allowed for the removal of random noise, cosmic ray events, dark current, and hot pixels. All these experiments were per- formed at least four times, and in each experiment 5–10 cells were re- corded and analyzed. Fluorescence microscopy Transfected cells in 35-mm glass coverslip culture dishes (Iwaki) were ob- served by confocal microscopy using a microscope (LSM510; Carl Zeiss MicroImaging, Inc.) with an F-FLUAR 40 /1.3 oil immersion objec- tive. Excitation of EGFP was performed using an argon ion laser at 488 nm. Emitted light was detected through a 505–550-nm bandpass filter from a 545-nm dichroic mirror. dsRed fluorescence was excited using a green he- lium-neon laser (543 nm) and was detected through both a 545-nm di- chroic mirror and a 560-nm long-pass filter. Data capture and extraction was carried out with LSM510 version 3 software (Carl Zeiss MicroImaging, Inc.) using tracking mode to exclude fluorescence cross talk. For the NF- B–EGFP fusion protein, mean fluorescence intensities were extracted for cells at each time point for both nucleus and cytoplasm. Nuclear/cytoplas- mic fluorescence intensity ratios were determined from these data. Con- comitantly, the mean cellular fluorescence intensities of I B -EGFP fusion proteins were extracted, and the fluorescence intensity relative to starting fluorescence was determined for each cell. All these experiments were performed at least five times, and each time four different fields, which each contained 3–5 transfected cells, were analyzed. Flow cytometric analysis After serum stimulation, Swiss 3T3 cells cultured in 25-cm2 flasks were de- tached as a single cell suspension with 900 l trypsin. The cells were stained by addition of 250 l of 50 g/ml propidium iodide, 0.15% Triton X-100, and 150 g/ml RNase A before analysis in an Altra flow cytometer (Beckman Coulter). Calcium measurement [Ca2 ]i levels in living 3T3 cells were monitored by fluorescence micros- copy using the Ca2 indicator Fluo-4. Cells in 35-mm glass coverslip cul- ture dishes (Iwaki) were incubated at 37 C for 20 min with the acetoxy- methyl ester of Fluo-4 (1 M Fluo-4-AM). After washing off the excess dye, Fluo-4–loaded cells were visualized with an F-Fluar 40 /1.3 oil im- mersion objective. It was observed by time-lapse fluorescence confocal microscopy that prolonged incubation with the fluorochrome led to a punctuate distribution of the dye within the cells. Flash photolysis 3T3 cells were loaded with the cell permeant acetoxy-methyl ester deriva- tive fluorescent indicator Fluo-4 (0.7 M) and the caged calcium com- pound nitro-phenyl-NP-EGTA (NP-EGTA; 0.7 M; Ellis-Davies and Kaplan, 1994) or Diazo-2 (Lannergren and Arner, 1992; Molecular Probes, Inc.). Photo-release of the caged compounds was induced by a train of 4- s flashes over a period of 10 s using 360-nm light from a micropoint photoac- tivation system (Photonic Instruments, Inc.) RNA extraction and RT-PCR Total RNA was extracted by using the RNeasy kit (QIAGEN), following the manufacturer’s instructions. 2 g of RNA was used to perform reverse tran- scriptase PCR (using 200 U M-MLV reverse transcriptase; Promega). For PCR amplification, 5 l of RT products were incubated with 20 pmol of spe- cific primers and 1 U of Taq DNA polymerase (Promega). PCR products were resolved by agarose gel electrophoresis and were visualized by stain- ing with ethidium bromide. The primers designed with primer 3 software (Rozen and Skaletsky, 2000) were as follows: CD1: 5’-ATGCTGGTTTTTGC- CTGAAG-3’, 5’-TTGTCCCCAATCTCCTTGTC-3’; cyclophilin A: 5’-GTG- GTCTTTGGGAAGGTGAA-3’, 5’-TTACAGGACATTGCGAGCAG-3’. Western blot analysis 20 g of total cell extract in sample buffer (62.5 mM Tris HCl, pH 6.8, 10% [vol/vol] glycerol, 1% SDS, 1% [vol/vol] mercaptoethanol, 0.1% bro- mophenol blue, and protease inhibitor cocktail [Sigma-Aldrich]) was loaded onto a 10% SDS-polyacrylamide gel. For nuclear extracts, cells cultured on 10-cm Petri dishes were washed once with PBS 1 , and scraped in 400 l of 0.3 M sucrose in AT buffer (AT buffer: 10 mM Tris HCl, pH 8.0, 3 mM CaCl2, 2 mM MgCl2, 0.5 mM DTT, 0.15% [vol/vol], and Triton X-100) and were carefully layered on 400 l 0.4 M sucrose in AT buffer. After centrifugation at 600 g (for 10 min at 4 C), the pellet was washed once with ice-cold pellet buffer (50 mM Tris HCl, pH 8.0, 1 mM Calcium-dependent NF- B activity during cell cycle | Sée et al. 671 DTT, 0.2 mM EDTA, and 0.5 mM PMSF) and resuspended in 20 l pellet buffer, before SDS-PAGE. A Bradford assay was performed before loading to determine the protein concentration. Polypeptides separated by SDS- PAGE were blotted onto a nitrocellulose membrane (0.22 m; Bio-Rad Laboratories). Blocking and antibody incubations were performed as al- ready described (See et al., 2001). The specific bands were detected by ECL (West Pico; Pierce Chemical Co.). Blots were exposed to BIOMAX-MR KODAK films. All Western blot analyses were performed at least three times. Online supplemental material Fig. S1 shows controls for photoactivation of the calcium chelator Diazo-2. Fig. S2 shows effects of CaMKII and calcineurin inhibitors on serum-induced NF- B–dependent transcription. Fig. S3 shows the effect of siRNA knock- down of p65 on serum-induced CD1 transcription. Fig. S4 shows the effect of BAPTA, PD98059, and wortmannin on p-IKK and p-I B levels and the effect of PD98059 on I B degradation and p65 translocation. Video 1 shows p65 translocation into the nucleus and I B degradation after 10% FCS stimulation. We gratefully acknowledge C. Wood for preliminary work and advice, E. Costello for mutant I B expression vector, N. Inohara for the IKK mutant expression vectors, and R. Pestell for the cyclin D1 promoter luciferase re- porters. We thank D. Fernig, S. Edwards, and H. Bito for critical reading of the manuscript. We thank the MRC, North West Cancer Research Fund, and HEFCE for funding this work. The project was also supported by Carl Zeiss MicroIm- aging, Inc. and the Hamamatsu Corporation. Submitted: 25 February 2004 Accepted: 7 July 2004 References Albanese, C., J. Johnson, G. Watanabe, N. Eklund, D. Vu, A. Arnold, and R.G. Pes- tell. 1995. 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