664 The Journal of Cell Biology | Volume 166, Number 5, 2004 Figure 2. The induction of CD1 promoter activity by serum is NF- B dependent. (A) Cells were transfected with the CD1 reporter vector. After a period of 24 h of serum starvation, cells were stimulated or not (ct) with 10% FCS in the absence (ct) or presence of an NF- B inhibitor (5 M Bay117082; 20 min preincubation), or were cotransfected with a truncated active mutant of I B . (B) Cells were cotransfected with the CD1 reporter vector and either a control empty vector, or a vector expressing either transdominant-negative IKK or IKK . (C) Schematic diagrams of the several CD1-luciferase reporters used. (D) Cells were transfected with the indicated reporters. (A, B, and D) Results are expressed as fold activation relative to the level measured in nonstimulated starved cells. Cells were assayed for luciferase activity 6 h after serum stimulation. Histograms are means SEM of triplicate values. Each experiment was performed three times. **, (P 0.05) indicates statistically significant difference (two-tailed t test). calcium increase, based on photoactivation of the caged cal- cium scavenger Diazo-2, which displays a low affinity for Ca2 before photolysis. This affinity is increased 30-fold af- ter illumination at 360 nm. Diazo-2 was loaded into 3T3 cells that were then subjected to a brief (10 s) period of pho- toactivation. This photoactivation alone induced a transient 20% decrease in [Ca2 ]i levels (Fig. 5 A). No significant de- crease in [Ca2 ]i occurred in cells that were loaded with Di- azo-2 but were not illuminated, nor in cells that were illumi- nated but not loaded with Diazo-2 (unpublished data). Experiments where illumination was performed immediately after serum induction showed that the photolysis of Diazo-2 inhibited the serum-induced calcium peak (Fig. 5 B), but had no effect on subsequent intracellular calcium levels. Pho- toactivation of Diazo-2 after serum stimulation also blocked detectable p65 translocation into the nucleus (Fig. 5 C) as well as NF- B–dependent gene transcription from the NF- B consensus and CD1 promoters (threefold decrease; Fig. 5 D). For all these experiments, the control cells shown were the nonilluminated fields from the same illuminated experi- ment. Additional controls (analyzing the effects of serum stimulation on illuminated cells that were not previously loaded with Diazo-2) were also performed to ensure that the lack of translocation and transcription was not due to illumi- nation, UV-toxicity, or free radical production. These con- trol experiments gave identical results to the nonilluminated cells with respect to p65 translocation and reporter lumines- cence (unpublished data). Additionally, we also analyzed the effects of serum on cells that were loaded with Diazo-2 and subjected to photoactivation 5 min before serum stimulation (Fig. S1, available at http://www.jcb.org/cgi/content/full/ jcb.200402136/DC1). In this important control experi- ment, the calcium response of the cells to the serum stimula- tion was identical to the usual response that was obtained in the absence of previous uncaging. Furthermore, the NF- B translocation and NF- B–Luc responses to serum were unaf- fected by the uncaging procedure (Fig. S1). To further characterize the relationship between the tran- sient [Ca2 ]i increase and subsequent p65 activation, we per- formed the same experiment using flash photolysis with a