Figure 2.

EGF triggers Ca²⁺ release by nuclear InsP₃ and nuclear PI(4,5)P₂ hydrolysis. A, confocal images of SKHep-1 cells loaded with Fluo-4/AM expressing cytosolic InsP₃-buffer, nuclear InsP₃-buffer, or mRFP (control). Images show cells before EGF stimulus (baseline) and the peak (15 min) of EGF-induced Fluo-4/AM fluorescence intensity changes. Expression of constructs was checked by detection of mRFP. Bar = 10 μm. B, bar graph showing Fluo-4/AM fluorescence intensity peak in cytosolic and nuclear regions of control, cytosolic, or nuclear InsP₃-buffer expressing cells upon EGF stimulus (8–11 cells in each group: *, p < 0.05; **, p < 0.01; and ***, p < 0.001) (cytosol: one-way ANOVA, F(2,24) = 17.17, p < 0.0001; nuclear, one-way ANOVA, F(2,26) = 28.03; p < 0.0001.) C, average traces of SKHep-1 expressing cytosolic or nuclear InsP₃-buffer or control cells stimulated with EGF. Cytosolic or nuclear regions for fluorescent intensity measurements were selected using the assistance of the digital image of contrast (D.I.C) images, see squares at the nucleus and non-nuclear regions. Nuclear but not cytosolic InsP₃-buffer blocked the peak of EGF response in both compartments. D, bar graph represents the amount of PI(4,5)P₂ of nucleus isolated from control or EGF-stimulated hepatocytes (n = 6). 5 min of EGF stimulation reduces nuclear PI(4,5)P₂ by 64 ± 1.5% (p < 0.05) (Student's t test).