Epidermal growth factor (EGF) triggers nuclear calcium signaling through the intranuclear phospholipase Cδ-4 (PLCδ4)

Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca2+ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within the nucleus, leading to Ca2+ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P2 hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca2+ signaling and downstream events. We found that EGF-induced Ca2+ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca2+ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP3) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P2 by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP3 modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.

The spatial-temporal distribution of calcium (Ca 2ϩ ) signals contributes to the versatility of this second messenger (1). For example, increases in Ca 2ϩ within the cell nucleus selectively promote cellular events such as gene transcription (2), cell proliferation, and tumor growth (3). Moreover, nuclear Ca 2ϩ signals can be regulated independently of cytosolic Ca 2ϩ signals (4). This is possible because the nucleus contains the machinery necessary for Ca 2ϩ mobilization (5)(6)(7)(8). Specifically, the nuclear envelope is contiguous with the endoplasmic reticulum (ER) 3 (9) and has invaginations that can reach deep within the nucleoplasm (10). These invaginations express InsP 3 Rs, store and release Ca 2ϩ in an InsP 3 -sensitive fashion (5,11), and have been referred to as the nucleoplasmic reticulum (NR) (5,10). The distribution of InsP 3 R isoforms and also Ca 2ϩ signaling in the nucleus become altered in various disease states, such as fatty liver disease (12) and cholangiocarcinoma (13). Intranuclear InsP 3 is formed from PI(4,5)P 2 hydrolysis, which is induced by growth factors such as hepatocyte growth factor (7) and insulin (8). Some RTKs undergo nuclear translocation upon activation, which appears necessary for initiation of nuclear Ca 2ϩ signals (7,8), so this could be one mechanism to regulate Ca 2ϩ release locally.
Nuclear localization of EGFR is of clinical relevance, as it is correlated with cancer prognosis (14) and relates to resistance to various cancer therapies (15,16). Nuclear EGFR function has been the subject of extensive investigation, as has been nuclear targets of EGFR. EGFR can shuttle from the cell surface to the nucleus (17,18) where it acts as a transcriptional regulator (19,20), transmits signals (21,22), and is involved in processes such as cell proliferation, tumor progression, and DNA repair and replication (23). However, it is unknown whether EGFR is linked to nuclear calcium release.
PLC could be involved in primary nuclear Ca 2ϩ signaling because activation of InsP 3 receptors generally is involved in the release of Ca 2ϩ in the nucleus (24). In mammals, the PLC family is composed of 13 isozymes, divided into six classes as follows: ␤, ␥, ␦, ⑀, , and , according to their structures (25). All these isozymes catalyze the reaction of PI(4,5)P 2 cleavage, generating InsP 3 and diacylglycerol (DAG), but each one possesses unique physiological functions (25). Some PLCs are described within the nucleus in specific cell types such as PLC␤1, PLC␥1, PLC␦1, PLC␦4, and PLC (26,27). The activity of these nuclear PLCs is related to the regulation of several cellular processes, such as proliferation and differentiation. These particular isoforms also have been linked to specific diseases, including myelodysplastic syndromes (PLC␤1), neurological diseases (PLC␥1), and infertility (PLC and PLC␦1-4) (28).
PLC␦4 has been identified as a nuclear protein in different cell types and may be involved in proliferative processes (27,(29)(30)(31). It was first purified from regenerating rat liver protein extracts (32), and its gene was cloned (33) from a regenerating rat liver cDNA library, indicating a possible role of PLC␦4 in cell proliferation. Despite its nuclear localization, it is unknown how human PLC␦4 is activated within the nucleus. This work investigates whether EGFR activates nuclear Ca 2ϩ signaling via PLC␦4.

EGFR translocates to the nucleus and induces Ca 2؉ signals
Stimulation with EGF induces its RTK EGFR to translocate to the nucleus (18), similar to what has been shown for other RTKs, including the hepatocyte growth factor (HGF) receptor, c-Met, and the insulin receptor (7,8). EGFR translocated to the nucleus in both primary rat hepatocytes and SKHep-1 cells, a human liver cancer cell line (Fig. 1A). EGFR protein levels in the nuclear fraction were higher than in nonstimulated control cells as soon as 2.5 min after stimulation with EGF (Fig. 1A). In SKHep-1 cells, EGFR decreased in the non-nuclear fraction as it increased in the nuclear fraction. The peak of EGFR nuclear translocation in this cell line was at 10 min (Fig. 1A). Super-resolution immunofluorescence showed that EGFR accumulated in the nucleus within 10 min of exposure to EGF (Fig. 1B). EGF stimulation induced bigger EGFR clusters throughout the cells, including at the nucleus, as demonstrated previously (15). These data demonstrate that EGF induces EGFR to translocate to the nucleus.
Translocation of EGFR to the nucleus depends on clathrin-mediated endocytosis (17), so we used clathrin heavy chain 2 (CHC2) siRNAs to disrupt this endocytic pathway and thereby inhibit internalization of EGFR. Fig. 1C shows that siRNA treatment reduced CHC2 expression by 94 Ϯ 3% using the CHC2 siRNA 1 and by 87 Ϯ 8% using the CHC2 siRNA 2, relative to control (p Ͻ 0.001). Stimulation of control cells with EGF led to a gradual Ca 2ϩ increase with some superimposed oscillations, similar to the Ca 2ϩ signal pattern induced by other growth factors (7,8), but CHC2 knockdown diminished the peak of EGF-induced Ca 2ϩ signals by 85 Ϯ 2% (p Ͻ 0.001) using the CHCH2 siRNA 1 and by 100 Ϯ 2% (p Ͻ 0.001) using the CHCH2 siRNA 2, compared with control (Fig. 1D). This indicates that most of the Ca 2ϩ signals induced by EGF depend on EGFR internalization.

EGF triggers intranuclear PI(4,5)P 2 hydrolysis and InsP 3 formation
EGF induces intracellular Ca 2ϩ transients through InsP 3 (34), so we investigated whether EGF-induced InsP 3 formation in the nucleus was responsible for increasing intranuclear Ca 2ϩ . SKHep-1 cells were transfected with cytosolic or nuclear InsP 3 -buffer constructs whose targeting was confirmed by subcellular localization of the monomeric red fluorescent protein (mRFP) ( Fig. 2A). Fluo-4/AM was used to monitor Ca 2ϩ release in response to EGF. Cytosolic InsP 3 -buffer decreased the Ca 2ϩ response by 50.5 Ϯ 6.1% in the nucleus and by 53.8 Ϯ 4.5% in the cytoplasm (Fig. 2B), but EGF-induced Ca 2ϩ signals were decreased by 86 Ϯ 2.7% in the nucleus and by 96 Ϯ 3.9% in the cytoplasm in cells expressing the nuclear InsP 3 -buffer (Fig. 2B). This suggests that EGF triggers nuclear Ca 2ϩ signals by inducing the formation of InsP 3 within the nucleus, similar to what has been observed in liver cells stimulated with HGF (7) or insulin (8). To investigate whether EGF induces nuclear PI(4,5)P 2 hydrolysis to produce InsP 3 , PI(4,5)P 2 was measured in nuclear fractions of hepatocytes. Nuclei from EGF-stimulated cells contained 64 Ϯ 1.5% less PI(4,5)P 2 than nuclei from control cells (Fig. 2D). These findings provide evidence that EGF triggers nuclear PI(4,5)P 2 hydrolysis and local InsP 3 production to generate Ca 2ϩ signals.

EGF stimulates intra-nuclear PKC activity
PI(4,5)P 2 hydrolysis generates not only InsP 3 but also DAG, which can activate PKC (24). Increases in nuclear DAG can either participate in translocation of PKCs, from the cytosol to the nucleus, or can directly activate PKCs that reside in the nucleus (35). To determine whether EGF triggers nuclear PKC activity, we used a Förster resonance energy transfer (FRET) reporter based on PKC activity and tagged to a nuclear localization signal (NucCKAR) (36). The nuclear localization of this construct was confirmed by intra-nuclear detection of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fluorescence by confocal microscopy (Fig. 3A). Phosphorylation of the reporter by PKC results in decreased rather than increased FRET (36), so the CFP/YFP ratio was monitored to reflect PKC activity. NucCKAR-expressing SKHep-1 cells were stimulated with EGF, phorbol 12-13-dibutyrate (PdBu) as a positive control for PKC activation, or arginine vasopressin (AVP) to reflect cytosolic activation of PKC, and then changes in FRET were followed for 30 min (Fig. 3B). PdBu increased CFP/YFP emission of NucCKAR (Fig. 3, B and C), and similar results were obtained with EGF treatment (Fig. 3, B and C), indicating that either stimulus increases nuclear PKC activity. Unlike EGF, stimulation with AVP, which activates a prototyp-

PLC␦4 mediates EGF-induced Ca 2؉ signals and cell proliferation
ical G-protein-coupled receptor in the plasma membrane, did not increase the CFP/YFP emission ratio of NucCKAR (Fig. 3, B and C). This result suggests that subplasmalemmal formation of DAG in response to AVP does not activate nuclear PKC. Treatment with the broad-range PKC inhibitor Gö6983 (Fig.  3D) decreased the EGF-induced CFP/YFP emission ratio, providing additional evidence that EGF triggers PKC activity within the nucleus.

PLC␥1 is localized to the cytosol and PLC␦4 is in the nucleus
PLC mediates PI(4,5)P 2 hydrolysis (28) and formation of InsP 3 and DAG (29,30), so the relative role played by several candidate PLC isoforms during EGF stimulation was investigated. PLC␥1 is the primary isoform typically thought to bind to and be activated by RTKs (1). PLC␦4 has been recognized as a predominantly nuclear PLC isoform, and its expression is increased in liver regeneration (33), so we compared the localization of these two isoforms in SKHep-1 cells. Confocal immunofluorescence showed that PLC␥1 is mostly localized to the cytosol, and this pattern does not change upon EGF stimulation (Fig. 4A). In contrast, PLC␦4 was detected exclusively in the nucleus of SKHep-1 cells, demonstrated by both confocal microscopy ( Fig. 4B) and Western blotting (data not shown). The subcellular localization of these PLC isoforms was also assessed in primary rat hepatocytes. Similar to what was observed in SKHep-1 cells, PLC␥1 was found in non-nuclear fractions of both control and EGF-stimulated cells, whereas PLC␦4 was only detected in the nuclear fractions (Fig. 4C). The amount of PLC␥1 or PLC␦4 in each cell fraction was not altered by stimulation with EGF ( Fig. 4C), indicating that there is no translocation of these PLCs between nuclear and non-nuclear compartments in response to EGF. Further examination showed that EGF stimulation for up to 10 min does not induce PLC␥1 to translocate from the cytoplasm to the nucleus (Fig. 4D).

PLC␦4 participates in cell cycle progression
Nuclear Ca 2ϩ is important for cell proliferation (3), an important downstream effect of EGF. Therefore, we investigated whether PLC␦4 is involved in EGFR-mediated cell proliferation. Cell proliferation as measured by either cell counting ( Fig. S1) or BrdU incorporation ( Fig. 6A) was decreased by over 67% in cells in which PLC␦4 expression was reduced. SKHep-1 proliferation was restored when PLC␦4 expression was recovered ( Fig. S1). Cell death does not mediate the decreased proliferation because annexin V-FITC and propidium iodide assays did not reveal cell death between control and siRNA-treated groups (Fig. 6B). Next, the role of PLC␦4 in cell cycle progression was investigated. To understand the basis for this, the effects of PLC␦4 on the expression of various cyclins were studied (Fig. S1). Expression of cyclin B1, a G 2 /M-phase checkpoint protein, and cyclin A, an S-phase checkpoint protein, was decreased in PLC␦4 knockdown cells compared with cells treated with control siRNA (Fig. 6C). A nuclear InsP 3 -buffer also decreased the expression of cyclins A and B. These findings suggest that nuclear PLC␦4 affects cell cycle progression, in part by affecting cyclin expression.

Discussion
EGFR has several direct effects on signaling in the nucleus, including interaction with transcription factors (37) and phosphorylation (21). This work extends this repertoire of intranuclear actions by showing that EGFR translocates to the nucleus of hepatic cells to initiate InsP 3 -mediated Ca 2ϩ signals. EGFR likely translocates from the plasma membrane to the nucleus via the ER (20,23), and a similar trafficking route has been

Figure 1. EGF induces EGFR nuclear translocation and clathrin-mediated endocytosis-dependent Ca 2؉ signals.
A, Western blot analysis of EGFR in non-nuclear and nuclear fractions isolated from resting (control) or EGF-stimulated hepatocyte and SKHep-1 cells. ␣-Tubulin and lamin B1 were used as purity controls for non-nuclear and nuclear fractions, respectively. Indicated kDa values represent the position of the referenced bands from pre-stained protein standard. Densitometric measurements of cellular fractions show that nuclear EGFR is maximal within 10 min of stimulation in SKHep-1 cells and within 2.5-5 min (p Ͻ 0.01) in hepatocytes when compared with the control (0 min). Values are scaled relative to the initial amount in the non-nuclear fraction and relative to the amount at 2.5 min in the nuclear fraction (mean Ϯ S.E.; n ϭ 3) (nuclear SKHep-1 fractions, one-way ANOVA, F(4,10) ϭ 3.215; p ϭ 0.0611; nuclear hepatocyte fractions, one-way ANOVA, F(4,10) ϭ 8.530, p ϭ 0.0029). B, gated STED super-resolution images of control SKHep-1 cells (right image) and 10 min after EGF stimulation (middle image). Serial optical sections were collected for three-dimensional reconstruction; y-z sections are shown at the right of each image. EGFR is represented in green; lamin B1 is in red, and the nuclear compartment marked in blue. Note that EGFR redistributes to the region of the nuclear envelope as well as within the nuclear interior (arrows). Bar ϭ 10 m. Scatter plot (right panel) showing the quantification of EGFR that co-localizes with the nucleus before and after EGF stimulation. n ϭ 7-12 cells for each group. The image collection settings for fluorescence quantification was adjusted according to the cells stimulated with EGF to avoid nuclear-saturated pixels of EGFR clusters. ***, p Ͻ 0.001 (Student's t test).

PLC␦4 mediates EGF-induced Ca 2؉ signals and cell proliferation
described for both viruses and toxins as well (38 -40). Trafficking of other RTKs from the plasma membrane to the nucleus has also been described. For example, c-Met that is biotinylated at the plasma membrane can be recovered from the nucleus after cells are stimulated with HGF (7). Similarly, fluorescentlylabeled EGF can be tracked from the cell membrane to the cell nucleus over a 10-min period (18). Furthermore, super-resolution imaging can be used to quantify the amount of EGF/EGFR clusters that accumulate in the nucleus (18). The peak in nuclear translocation of EGFR coincides with the peak in Ca 2ϩ signals (Fig. 1, A-C). This work builds on the previous observation that translocation of EGFR to the nucleus depends on dynamin and clathrin-mediated endocytosis (17) by showing that knockdown of clathrin reduces EGF-induced Ca 2ϩ signals. These findings are consistent with the idea that EGFR must translocate to the nucleus in order to initiate nuclear Ca 2ϩ signals, similar to what has been shown for c-Met (7).
Ca 2ϩ signaling in the nucleus rather than cytosol is important for proliferation and tumor growth (3). Nuclear Ca 2ϩ can also regulate gene expression (2, 4). Thus, increases in nuclear C, average traces of SKHep-1 expressing cytosolic or nuclear InsP 3 -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 InsP 3 -buffer blocked the peak of EGF response in both compartments. D, bar graph represents the amount of PI(4,5)P 2 of nucleus isolated from control or EGF-stimulated hepatocytes (n ϭ 6). 5 min of EGF stimulation reduces nuclear PI(4,5)P 2 by 64 Ϯ 1.5% (p Ͻ 0.05) (Student's t test).

PLC␦4 mediates EGF-induced Ca 2؉ signals and cell proliferation
Ca 2ϩ could be one mechanism for EGF to promote those cellular processes. The nucleus contains the machinery necessary for InsP 3 receptor-mediated Ca 2ϩ release (5,7,8,24). Although PLC-mediated PI(4,5)P 2 hydrolysis and formation of InsP 3 and DAG within the cytoplasm have been described extensively (1), there are fewer studies showing that PI(4,5)P 2 hydrolysis also occurs within the nucleus. This work provides evidence that EGF triggers PI(4,5)P 2 hydrolysis and InsP 3 formation within the nucleus (Figs. 2 and 5), similar to what has been observed in response to stimulation with HGF (7) or insulin (8).
PLC within the nucleus is involved in signal-transduction pathways that are separate from those activated by isoforms localized in other compartments (41). The most common par-adigm links PLC␤ isoforms with G-protein-coupled receptors, whereas RTKs typically are associated with PLC␥ isoforms (42). For example, the activated nuclear metabotropic glutamate 5 (mGlu5) receptor couples to G q/11 and a nuclear isoform, PLC␤1, to generate the InsP 3 -mediated release of Ca 2ϩ from Ca 2ϩ -release channels in the nucleus of primary striatal neurons (43). This work provides evidence that EGF induces hydrolysis of nuclear PI(4,5)P 2 by the intra-nuclear PLC␦4, rather than by PLC␥1 (Fig. 5). It remains unclear whether PLC␦4 is activated directly or indirectly, as a direct binding of PLC␦4 with nuclear EGFR could not be demonstrated by co-immunoprecipitation (data not shown).
There are a number of previous studies about the function of nuclear PLCs. Some studies suggest that PLC positively regulates the cell cycle. PLC␤1 can regulate insulin-like growth factor-1-stimulated DNA synthesis, and its overexpression increases proliferation in the absence of mitogenic stimuli (44,45). Furthermore, PLC␤1 in the nucleus regulates transcriptional levels of genes such as cyclin D3 and c-jun activation in skeletal muscle cells (45,46). This study demonstrates that PLC␦4 also plays a role in cellular proliferation and specifically in cell-cycle control. Knockdown of PLC␦4 leads to decreased cell proliferation without affecting apoptosis in SKHep-1 cells (Fig. 6). The cell cycle profile is accompanied by reduced expression of cyclins A and B1 (Fig. 6). Consistent with this, decreased cyclin B1 could interfere with the transition through the G 2 /M phase of the cell cycle. Furthermore, it was demonstrated that PLC␦4 knockdown impaired human mesenchymal stem/stromal cells proliferation, without inducing cell death (31).
The direct target of PLC signaling is typically PKC, which is activated by the PI(4,5)P 2 hydrolysis products DAG and InsP 3 (35). Because EGF activates PKC within the nucleus (Fig. 3), and several nuclear proteins are PKC substrates, this may also be relevant for processes such as gene expression and changes in chromatin structure (47). Activation of PKC may affect cell cycle progression as well (48,49). PKC␣ is necessary for PLC␤1mediated regulation of cyclin D3 and cell proliferation in human erythroleukemia cells (50), and PKC␣ regulates cyclin B1 leading to effects on G 2 /M transition. PKC inhibitors can decrease cyclin B1 expression consequent to PKC down-regulation (51). The increase in nuclear DAG level observed during the G 2 phase is sufficient to activate nuclear PKC␣/PKC␤. PKC phosphorylates lamin B1 at sites involved in mitotic disassembly of the nuclear lamina, one of the most critical events in the G 2 /M transition (52).
In light of these considerations, the current findings identify novel signaling events that may link EGFR nuclear translocation to stimulation of cell growth (Fig. 7). Activation of this signaling pathway may, in turn, be important for hepatocyte proliferation, especially because liver regeneration is absolutely dependent on the presence of either EGF or HGF (53). This is also consistent with the observation that PLC␦4 expression is increased in liver regeneration as well as in liver cancer cell lines (33,55). Furthermore, it is consistent with data from PLC␦4 knockout mice that show increases in bromodeoxyuridine (BrdU) are delayed after partial hepatectomy, although liver regeneration is not delayed (55). Therefore, strategies to selec-

PLC␦4 mediates EGF-induced Ca 2؉ signals and cell proliferation
tively inhibit PLC␦4 could be important for selective inhibition of liver cancer, which is the third leading cause of cancer death worldwide.
Hepatocytes were isolated from healthy male Sprague-Dawley rats (190 -200 g; Charles River Laboratories), as described previously (56). Primary cells were cultured at 37°C in 5% CO 2 in Williams' medium E (Life Technologies, Inc.) containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin and plated on collagen-coated coverslips (50 g/ml) (BD Biosciences). Hepatocytes were used within 24 h of isolation. All animal studies were approved by the Yale Univer-

Subcellular fractionation and Western blotting
Cells were washed twice with ice-cold 10 mM PBS, pH 7.4 (PBS) (Sigma), harvested by scraping, and lysed in a buffer with 20 mM HEPES, pH 7.0, 10 mM KCl, 2 mM MgCl 2 , 0.5% Nonidet P-40. After incubation on ice for 10 min, the cells were homogenized by vortex. The homogenate was centrifuged at 1500 ϫ g for 5 min to sediment the nuclei. The supernatant was then centrifuged at a maximum speed of 16,100 ϫ g for 20 min, and the resulting supernatant formed the non-nuclear fraction. The nuclear pellet was washed three times with lysis buffer to remove any contamination from cytoplasmic membranes. The isolated nuclei were resuspended in NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, 0.5% Nonidet P-40), and the mixture was sonicated briefly to aid nuclear lysis. Nuclear lysates were collected after centrifugation at 16,100 ϫ g for 20 min at 4°C. Protease and phosphatase inhibitors (Sigma) were added to all buffers. Proteins were quantified by Bradford assay (Sigma).

InsP 3 -buffer constructs and calcium imaging
Generation of InsP 3 -buffer constructs was as described previously (7). Briefly, the InsP 3 -binding domain (residues 224 -605) of the human type I InsP 3 receptor was tagged with an mRFP and with a nuclear localization signal or nuclear exclusion signal to generate the nuclear and cytosolic constructs, respectively. The adenoviruses were built by ViraQuest Inc. Cells were infected with 10 multiplicities of infection, and analysis was performed in 48 h. For calcium imaging, cells were incubated with 5 M Fluo-4/AM (Life Technologies, Inc.) and placed in a custom-made perfusion chamber for EGF stimulation. Images were collected with a Zeiss LSM 710 DUO NLO. Nuclear and cytosolic regions were selected with the assistance of the brightfield images.

Nuclear PI(4,5)P 2 extraction and detection
PI(4,5)P 2 from samples was detected by a mass ELISA kit (Echelon Biosciences, UT). 5 min after EGF stimulation, cell nuclei were isolated as described previously (7,8), washed three times with PBS, and followed by PI(4,5)P 2 extraction as recommended by the kit manufacturer. In summary, the protein was precipitated with 0.5 M TCA and followed by neutral lipid extraction using MeOH/CHCl 3 (2:1) and acidic lipid extracted with MeOH, CHCl 3 , 12 M HCl (80:40:1). Lipids collected from split organic phase were dried before analysis.

FRET-based PKC activity reporter
The NucCKAR construct was developed by Gallegos et al. (36) and obtained from Addgene (plasmid no. 14869). Cells were transfected using FuGENE 6 (Promega), and microscope studies were performed in 48 h. 200 nM PdBu (Sigma) was used as a positive control. 250 nM Gö6983 (Tocris Bioscience, Bristol, UK) was used as a PKC inhibitor to confirm the specificity of EGF response. Pre-stimulation baseline was performed with only DMSO before perfusion with PdBu and Gö6983 as a vehicle control. Cells were placed in a custom-made perfusion chamber for agonist stimulation. CFP and YFP emission intensities were collected using a Zeiss LSM 710 DUO NLO with a ϫ63, 1.4 NA objective lens. With this construct, increased PKC activity results in decreased rather than increased FRET (36). Therefore, CFP/YFP rather than YFP/CFP emission ratio was calculated to reflect PKC activity (36) and was normalized using Microsoft Excel software.

FRET-based InsP 3 reporter
The FRET-based InsP 3 -biosensor with nuclear localization signal (FIRE-1nuc) was developed by Kapoor et al. (54). Cells were transfected using FuGENE 6 (Promega), and microscopic studies were performed in 48 h. Cells were placed in a custommade perfusion chamber for agonist stimulation. CFP and YFP emission intensities were collected using a Zeiss LSM 710 DUO NLO with a ϫ63, 1.4 NA objective lens. YFP/CFP emission ratio was calculated and normalized using ZEN (Zeiss), Microsoft Excel, and GraphPad Prism software.

Proliferation assays
SKHEP-1 cells were seeded into 96-well plates (Corning, NY) at a density of 1 ϫ 10 4 and transfected with Lipofectamine RNAiMAX with control siRNAs, PLC␦4 siRNAs, and PLC␥1 siRNAs. The cells were transfected with 50 nM of each siRNA. Cells without transfection reagent and with Lipofectamine were used as a control group. The Cell Proliferation ELISA kit (Roche Applies Science, offered by Sigma) was used according to the manufacturer's instructions. Briefly, after 48 h, cells were cultivated with BrdU (0.2 M) for 2 h in serum-free media, fixated for 30 min, and incubated with anti-BrdU-POD for 90 min at room temperature. After three washing steps, 3,3Ј,5,5Ј-Tetramethylbenzidine was added, and the plate was read at 370 and 492 nm (Synergy 2, Biotek).
For proliferation curve assay, SKHep-1 cells were transfected with control siRNA 1 or PLC␦4 siRNA 1. After 48 h, cells were collected by trypsinization and counted in a Neubauer chamber using trypan blue for viability exclusion in triplicate.
For the proliferation recovery experiment, the Tet-pLKOneo plasmid (Addgene ID. 21916) was used to express shRNAs under the control of a doxycycline promoter. The PLCD4 shRNAs target sequences were obtained from "The RNAi Consortium Collection" (https://portals.broadinstitute.org/gpp/ public/), 4 and the shRNAs were designed and cloned according to Addgene instructions. In summary, shRNA sequences were subcloned into Tet-pLKO-neo after digestion with AgeI and EcoRI. We used the following target sequences for the shRNAs: PLC␦4 shRNA 1 (5Ј-AGAGCAGCGTCGAGGGATATA-3Ј); PLC␦4 shRNA 2 (5Ј-ACTACCACTTCTACGAGATAT-3Ј); Figure 7. Proposed model: EGF triggers nuclear PI(4,5)P 2 hydrolysis, local InsP 3 production, and Ca 2؉ release into the nucleoplasm through intra-nuclear PLC␦4, which mediates cell proliferation. Scheme of the proposed EGFinduced nuclear Ca 2ϩ signaling pathway. Upon EGF binding EGFR is activated and initiates signal transducer cascades. EGFR is shuttled to the nucleus to induce downstream targets. PLC␦4 is a nuclear enzyme induced by EGF stimulus and leads to PI(4,5)P 2 hydrolysis that generates InsP 3 and DAG. InsP 3 binds to InsP 3 R present on the inner nuclear envelope triggering Ca 2ϩ release in the nucleoplasm. DAG can activate nuclear-resident PKCs. Ca 2ϩ and PKCs can activate downstream targets which ultimately result in cell proliferation.

Cell death assay
For this experiment, the cells were incubated with 10 g/ml doxorubicin for 24 h, as a positive control for cell death. The cells were incubated with annexin V-FITC (Life Technologies, Inc.) and propidium iodide (Life Technologies, Inc.) for 1 h. Images were collected with a ϫ10 and ϫ20 objective lens on an Olympus IX70 inverted fluorescence microscope (Olympus America, Melville, NY), and the data obtained were quantified by counting cell labeled for annexin V-FITC, propidium iodide, double-labeled, and crossed with bright-field images used to obtain total cell count. Each treatment was made in triplicate, and three different images were counted, and the means were used for statistical analysis.

Statistical analysis
The significance of changes in treatment groups was determined by Student's t test or one-way analysis of variance with Tukey's multiple comparison tests if not otherwise stated, using GraphPad Prism software. Data are represented as mean Ϯ S.E.