Low Electric Treatment activates Rho GTPase via Heat Shock Protein 90 and Protein Kinase C for Intracellular Delivery of siRNA

Low electric treatment (LET) promotes intracellular delivery of naked siRNA by altering cellular physiology. However, which signaling molecules and cellular events contribute to LET-mediated siRNA uptake are unclear. Here, we used isobaric tags in relative and absolute quantification (iTRAQ) proteomic analysis to identify changes in the levels of phosphorylated proteins that occur during cellular uptake of siRNA promoted by LET. iTRAQ analysis revealed that heat shock protein 90 (Hsp90)α and myristoylated alanine-rich C-kinase substrate (Marcks) were highly phosphorylated following LET of NIH 3T3 cells, but not untreated cells. Furthermore, the levels of phosphorylated Hsp90α and protein kinase C (PKC)γ were increased by LET both with siRNA and liposomes having various physicochemical properties used as model macromolecules, suggesting that PKCγ activated partly by Ca2+ influx as well as Hsp90 chaperone function were involved in LET-mediated cellular siRNA uptake. Furthermore, LET with siRNA induced activation of Rho GTPase via Hsp90 and PKC, which could contribute to cellular siRNA uptake accompanied by actin cytoskeleton remodeling. Collectively, our results suggested that LET-induced Rho GTPase activation via Hsp90 and PKC would participate in actin-dependent cellular uptake of siRNA.

cellular uptake efficacy of nucleic acids between LET and electroporation is needed to be directly compared, LET without cytotoxicity is expected to be more useful drug delivery methods than electroporation. Alternatively LET can nonetheless deliver naked siRNA into targeted cells without the cytotoxicity that can accompany electroporation 19 . Moreover, we previously reported that various functional nucleic acids including siRNA delivered by LET have their functionality in the cells 19,20 . However, the detailed mechanism involved in cellular uptake of siRNA following LET remains unclear. The cellular uptake of naked siRNA promoted by LET is accompanied by changes in membrane potential mediated by transient receptor potential (TRP) channels 19 that facilitate cellular influx of cationic ions such as calcium ions in response to extracellular stimuli 21 . We previously showed that protein kinase C (PKC) is activated through influx of calcium ions into cells during transdermal delivery of cationic liposomes by LET 22 . PKC-associated signal transduction contributes to cellular uptake of macromolecules via actin remodeling [23][24][25] and calcium ions act as second messengers that meditate various signal transduction and cellular events for cellular uptake of macromolecules 26,27 . Thus, LET could induce changes in cellular physiology that result in activation of various signal transduction pathways. Furthermore, changes in the phosphorylation state of proteins involved in such pathways could affect the efficiency of cellular siRNA uptake.
To clarify the cellular uptake mechanisms involved in LET, in the current study we used isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to identify proteins that have altered phosphorylation status following LET. iTRAQ is a comprehensive protein analysis technique that involves binding of up to eight types of isobaric tags to peptides and subsequent MS/MS analysis to allow quantitative comparisons of the identified proteins 28 . Similar approaches are widely used in a variety of settings, including identification of serum biomarkers for lung cancer, metabolic analyses in heart mitochondria and signaling pathway analyses in the endoplasmic reticulum [29][30][31] . In this study we also show that the proteins showing up-regulated phosphorylation following LET promote actin cytoskeleton remodeling via activation of Rho GTPase that is associated with cellular siRNA uptake.

Results
iTRAQ analysis of phosphorylated proteins upregulated by LET. To identify proteins with altered phosphorylation status following LET, we performed an iTRAQ analysis of NIH 3T3 cells treated with LET. In this study, thresholds for detecting proteins with altered phosphorylation levels following LET were: 1.2 foldchange in levels of phosphorylated protein levels in LET-treated cells compared to untreated cells, and Unused Prot Score >0.62 that is indicative of the reliability of the identified proteins. Here, the number of proteins showing upregulated and downregulated phosphorylation following LET was 139 and 15, respectively (Fig. 1a). We next classified the proteins showing upregulated phosphorylation using a GO slim analysis to understand the biological function of the proteins (Fig. 1b). We previously reported that LET promotes cellular uptake of siRNA via an energy-dependent endocytic pathway 19 . One protein associated with vesicle-mediated transport in biological processes is Ras-related protein Rab-10 (Rab10) that has GTPase activity and is a key regulator of intracellular membrane trafficking 32,33 . Here we found that the level of phosphorylated Rab10 was 1.312-fold higher in cells exposed to LET relative to untreated cells. The 15 proteins that had the largest upregulation in phosphorylation following LET were: filamin C (Flnc), isoform 5 of heterogeneous nuclear ribonucleoproteins C1/C2 (Hnrnpc), protein Njmu-R(5730455P16Rik), 28 kDa heat-and acid-stable phosphoprotein (Pdap1), protein LYRIC (Mtdh), high mobility group protein B1 (Hmgb1), myristoylated alanine-rich C-kinase substrate (Marcks), elongation factor 1-β (Eef1b2), AHNAK nucleoprotein isoform 1 (Ahnak), heat shock protein HSP 90-α (Hsp90aa1 or Hsp90α), stress-70 protein, mitochondrial (Hspa9), poliovirus receptor (Pvr), Isoform 1 of E3 ubiquitin-protein ligase HUWE1 (Huwe1), protein AHNAK2 (Ahnak2) and pumilio 2 (Pum2) ( Table 1). Given the challenges in using GO slim analysis of GO term-attached proteins to identify proteins that contribute to intracellular delivery of macromolecules (Supplemental Table S1), we used STRING analysis of the top 15 phosphorylated proteins to characterize interactions between them. Hsp90aa1 (Hsp90α) interacted with the highest number of proteins, including Hnrnpc, Hmgb1, Marcks, Eef1b2 and Huwe1 (Fig. 1c). Although previous reports suggested that Hsp90 is involved in vesicular transport 34 , whether LET-induced Hsp90 phosphorylation contributes to cellular uptake of siRNA was unclear. Cellular uptake of siRNA and various macromolecules via Hsp90α activation induced by LET. We next examined how changes in Hsp90α phosphorylation status affected uptake of siRNA and macromolecules having different physicochemical properties such as liposomes. iTRAQ analysis revealed that Hsp90α phosphorylation was upregulated only in cells exposed to LET and not untreated cells (Table 1). Western blotting with UV-treated cells as a positive control for Hsp90α phosphorylation showed that the level of phosphorylated Hsp90α (Thr 5/7 ) increased by over 2-fold for both LET with and without siRNA relative to untreated control cells (Fig. 2a,b), suggesting that the presence of siRNA did not influence Hsp90α phosphorylation induced by LET. siRNA is a negatively charged, hydrophilic macromolecule 3 . To further understand how the surface charge and hydration of macromolecules affected LET-induced Hsp90α phosphorylation, cells were exposed to LET in the presence of cationic liposomes (cationic-lipo) and anionic liposomes (anionic-lipo) as charged macromolecules, and polyethylene glycol-modified liposomes (PEG-lipo) that have a hydrated surface layer. The levels of phosphorylated Hsp90α seen following LET in the presence of various macromolecules were similar to those obtained for LET both with and without siRNA (Fig. 2a,b), suggesting that the physicochemical properties of siRNA did not affect LET-induced Hsp90α phosphorylation.
To determine whether Hsp90α participates in LET-mediated cellular uptake of siRNA, we investigated the effect of the Hsp90 inhibitor 17-DMAG on cellular uptake of various macromolecules. The cellular uptake of various macromolecules was significantly enhanced by LET (Fig. 2c-f), and the cellular uptake of siRNA following LET was over 30 times higher than that of cells treated with siRNA alone (Fig. 2f), which was the highest seen among the macromolecules examined in this study. Furthermore, co-treatment with 17-DMAG inhibited www.nature.com/scientificreports www.nature.com/scientificreports/ LET-mediated cellular uptake of siRNA as well as anionic-lipo (Fig. 2d,f). These results suggested that Hsp90 activation contributed to LET-mediated cellular uptake of siRNA as well as anionic-lipo.
To further understand the siRNA cellular uptake mechanism via Hsp90 activation, we examined the effect of co-treatment of Hsp90 inhibitor 17-DMAG and Hsp70 inhibitor VER-155008 on the cellular uptake of siRNA. It is known that the inactivated Hsp90 is recovered by Hsp70 35 . If the inhibitory effect of 17-DMAG on the LET-mediated cellular uptake of will be attenuated by Hsp70, co-treatment of Hsp70 inhibitor VER-155008 with 17-DMAG will be more effective. As shown in Fig. 2f, co-treatment of VER-155008 with 17-DMAG prevented www.nature.com/scientificreports www.nature.com/scientificreports/ LET-mediated cellular uptake by over 80%, and the inhibitory effect was more potent that treatment of only 17-DMAG. This result suggested that Hsp90 activation was involved in LET-mediated cellular uptake of siRNA.
Involvement of PKC phosphorylation following LET-mediated Ca 2+ influx in cellular uptake of siRNA. In the iTRAQ analysis conducted here, levels of phosphorylated Marcks, which is a substrate for many PKC isoforms 36 , increased by over 2-fold in cells exposed to LET relative to untreated cells (Table 1). This finding suggests that PKC could also be a candidate signaling molecule that is affected by LET-mediated cellular uptake of macromolecules such as siRNA. Here, we first investigated whether PKCγ is phosphorylated by LET in the presence and absence of various macromolecules. Western blotting analysis with anti-phospho PKCγ (Thr 514 ) antibody indicated that the level of phosphorylated PKCγ increased by 3-fold following LET, and these levels were not affected by the presence of siRNA or other macromolecules (Fig. 3a,b). Next, to clarify the mechanism of PKCγ phosphorylation, we used fluo-4 fluorescent dye to examine Ca 2+ influx into the cells immediately after LET with or without various macromolecules. The fluorescent signals were equivalent to the amount of intracellular Ca 2+ induced by LET either with or without various macromolecules, whereas the enhanced fluorescent signals following LET alone were abolished by pretreatment with the Ca 2+ chelator EGTA (Fig. 3c). Increases in PKCγ phosphorylation following LET in the presence of siRNA were prevented by EGTA pretreatment (Fig. 3d,e), suggesting that Ca 2+ inflow contributed to the PKCγ phosphorylation induced by LET with siRNA. Moreover, EGTA pretreatment also inhibited LET-dependent enhancement in cellular uptake of siRNA (Fig. 3f). Together these results suggested that LET induced Ca 2+ influx into cells regardless of the presence of macromolecules having different physicochemical properties and that LET enhanced the cellular uptake of siRNA through PKCγ activation induced by Ca 2+ influx. However, the phosphorylation of PKCγ induced by LET/siRNA was not completely inhibited by blocking Ca 2+ inflow, while the statistical significance was observed. Moreover, the cellular uptake of siRNA was not effectively inhibited by the treatment of EGTA, being correlated with the insufficient inhibition of PKCγ phosphorylation (Fig. 3f). It is known that PKCγ is phosphorylated by diacylglycerol as well as Ca 2+ 37 . To clarify whether PKCγ is activated by except Ca 2+ inflow in the cells treated with LET/siRNA, we examined the effect of other PKC inhibitor Gö6983 that competitively inhibits ATP binding to the catalytic domain of PKC 37 . As shown in Supplementary Fig. S5, LET-mediated cellular uptake of siRNA was more potently inhibited by the treatment of Gö6983 in comparison with EGTA, suggesting that LET-induced Ca 2+ inflow-mediated is partly involved in the cellular uptake of siRNA via PKCγ activation.
Next, we examined the effect of Ca 2+ influx-mediated PKCγ activation on Hsp90α phosphorylation in cells exposed to LET in the presence of siRNA. As mentioned above, STRING analysis indicated that Hsp90α interacts with the PKC substrate Marcks, but whether Hsp90α and PKCγ interact was unclear. The level of phosphorylated Hsp90α in cells exposed to LET and siRNA was not affected by pretreatment with either EGTA or the PKC inhibitor chelerythrine, suggesting that Hsp90α phosphorylation associated with LET involves a pathway that is independent of PKCγ activated by Ca 2+ inflow ( Supplementary Fig. 4a,b). These results indicate that LET-mediated cellular uptake of siRNA could be differentially enhanced by Hsp90α and PKCγ activation.
In addition to NIH 3T3 cells, the LET-mediated cellular uptake of siRNA in mouse melanoma cell line B16-F1 was also inhibited by the treatment of Hsp90 inhibitor, calcium chelator or PKC inhibitor ( Supplementary Fig. 6). It is suggested that the involvement of these signaling molecules in LET-mediated cellular uptake is not specific to NIH3T3 cells, although further investigations are needed.
Effect of LET with siRNA on actin cytoskeleton remodeling. To understand cellular events activated by Hsp90α or PKCγ in LET-mediated cellular uptake of siRNA, we focused on the actin cytoskeleton that is www.nature.com/scientificreports www.nature.com/scientificreports/ linked to the plasma membrane 38 . Actin cytoskeleton remodeling is an essential step in endocytosis and is activated by various signaling molecules, including Hsp90 and PKC 23,39 . Staining of cells exposed to LET in the presence of siRNA with rhodamine-labeled phalloidin, which binds F-actin, yielded fluorescent signals from fiber-like structures observed at the neighboring plasma membranes as well as in the cytoplasm, whereas for cells exposed to LET alone, the fluorescent signals were limited to the cytoplasm (Fig. 4). These results indicated that LET with siRNA induced actin cytoskeleton remodeling that involved formation of lamellipodia and stress fibers associated with polymerization of cellular actin. The actin cytoskeleton remodeling induced by LET with siRNA could be prevented by pretreatment of EGTA, chelerythrine or 17-DMAG (Fig. 4), consistent with the results showing that these inhibitors prevented LET-mediated cellular uptake of siRNA (Figs 2f and 3f, and Supplementary Fig. 6). Together with previous studies showing that intracellular actin cytoskeleton remodeling participates in cellular uptake and vesicle transport 38 , actin cytoskeleton remodeling promoted by Hsp90α and PKCγ activation would be an essential cellular event for LET-mediated cellular uptake of siRNA.
Involvement of Rho GTPase in LET-mediated cellular uptake of siRNA. Rho GTPases are a major regulator of actin remodeling 40 . To investigate whether LET activates Rho GTPase, we measured Rho GTPase activity in cells exposed to LET alone or LET with siRNA. Rho GTPase activity was significantly increased after exposure to LET with siRNA (Fig. 5a). This increased activity was prevented by pretreatment with EGTA, www.nature.com/scientificreports www.nature.com/scientificreports/ chelerythrine or 17-DMAG, suggesting that LET with siRNA activated Rho GTPase via Hsp90 or PKC. Moreover, pretreatment with Rho inhibitor I prevented both actin remodeling seen following LET with siRNA treatment and LET-mediated cellular uptake of siRNA (Fig. 5b,c). These results suggested that LET-mediated cellular uptake of siRNA accompanied by actin remodeling was enhanced by Rho GTPase activity promoted by Hsp90 or PKC.

Discussion
We previously showed that LET promotes the cellular uptake of siRNA 19 . Moreover, LET has been used for the transdermal delivery of nanoparticles such as gold and poly(lactic-co-glycolic acid) (PLGA) nanoparticles 41,42 . In the present study, we examined changes in the phosphorylation status of cellular proteins to characterize the molecular mechanism underlying cellular uptake of siRNA mediated by LET. Exposure of NIH 3T3 cells to LET induced phosphorylation of various proteins that have different functions and participate in a range of biological processes (Fig. 1a,b). Among the 15 proteins that had the largest change in phosphorylation status, Hsp90α www.nature.com/scientificreports www.nature.com/scientificreports/ overlapped with the most cellular functions (Fig. 1c). Hsp90 functions as an ATP-dependent essential molecular chaperone in cells 43 . Post-translation modifications of Hsp90, including phosphorylation, induce structural rearrangements that promote ATP binding and subsequent hydrolysis, which in turn increase Hsp90 chaperone activity that facilitates signal transduction, protein folding and degradation, maturation of client proteins and vesicle transport 34,44 . Here we showed that the level of Hsp90α phosphorylated at Thr 5/7 was up-regulated by LET, similar to changes induced by UV treatment (Fig. 2a,b). Thr 5/7 of Hsp90α is located in the N-terminal domain, and phosphorylation of this region can determine the open and closed status that modulates both ATP hydrolysis and chaperone activity 43 . Although Thr 5/7 of Hsp90α is phosphorylated by DNA activated protein kinase (DNA-PK) 44 , this kinase was not among the 139 up-regulated phosphoproteins detected by iTRAQ analysis. However, the level of phosphorylated nuclear ubiquitous casein and cyclin-dependent kinases substrate (Nucks1), which are also DNA-PK substrates 45 , in LET exposed cells was 1.525 times higher than that in non-treated cells (Fig. 1a), suggesting that Hsp90α might be phosphorylated at least in part DNA-PK. The levels of phosphorylated Hsp90α did not change in the presence of siRNA or liposomes having different physicochemical properties such as surface charge or hydration layer thickness (Fig. 2a,b), suggesting that the presence of exogenous macromolecules did not affect LET-induced Hsp90α phosphorylation. Indeed, LET promoted the cellular uptake of not only siRNA but also cationic-lipo, anionic-lipo and PEG-lipo (Fig. 2c-f). Only cationic-lipo were taken up as efficiently by untreated cells, likely due to electrostatic interactions with cellular membranes. 17-DMAG is a geldanamycin analogue that competes with ATP for Hsp90 binding, resulting in inhibition of chaperone activity and cellular events that require Hsp90 activation 46 . Pretreatment of cells with 17-DMAG prevented the cellular uptake of anionic-lipo, PEG-lipo and siRNA, suggesting that Hsp90α activity contributed to LET-mediated cellular uptake of macromolecules (Fig. 2d-f). In contrast, 17-DMAG pretreatment did not affect LET-mediated cellular uptake of cationic-lipo (Fig. 2c), indicating that Hsp90 likely does not affect electrostatic interactions of macromolecules. Although Hsp90 was previously shown to regulate intracellular vesicle transport 34 , to our knowledge this is the first report on the involvement of Hsp90 in uptake of macromolecules and more investigations are needed to characterize the role of Hsp90 in this process.
We previously reported that LET facilitates the cellular uptake of siRNA via endocytosis, and that the cellular uptake could be prevented by the non-selective cationic ion channel blocker SKF96365 19 . Here we showed that calcium influx and levels of phosphorylated PKC-γ increased in LET-exposed cells both in the absence and presence of siRNA and various liposomes (Fig. 3a,b). Thus, similar to its effect on the Hsp90 phosphorylation status, LET could also induce PKCγ phosphorylation regardless of the presence of macromolecules. The iTRAQ analysis showed that levels of phosphorylated Marcks, a PKC substrate, increased by over 2-fold in LET-exposed cells, indicating that LET induced PKC activation. Ca 2+ chelation significantly prevented not only PKCγ phosphorylation but also cellular uptake of siRNA (Fig. 3d-f), suggesting that PKC activation following calcium influx contributes to LET-mediated cellular uptake of siRNA.
A study by Lu X. et al. proposed a synergistic interaction between PKCγ and Hsp90α wherein Hsp90α forms a chaperone complex with PKCγ that in turn promotes its translocation from the cytosol to the plasma membrane www.nature.com/scientificreports www.nature.com/scientificreports/ and subsequent activation 47 . At the membrane, Hsp90α Thr 115 , Thr 425 and Thr 603 residues are specifically phosphorylated by PKCγ, followed by a decrease in Hsp90 chaperone activity upon dissociation of PKCγ from Hsp90 in the cytoplasm. However, we showed that LET-mediated cellular uptake of siRNA could be prevented by pretreatment with 17-DMAG (Fig. 2b,f), suggesting that phosphorylated Hsp90α chaperone activity was present during PKCγ activation. Moreover, pretreatment with EGTA or the PKC inhibitor chelerythrine did not inhibit Hsp90α Thr 5/7 phosphorylation induced by LET with siRNA ( Supplementary Fig. S4a,b). Although further investigations are needed, PKCγ might not be the upstream signaling molecule for Thr 5/7 phosphorylation of Hsp90α in LET-mediated cellular uptake of siRNA and PKCγ and Hsp90α would differentially facilitate LET-mediated cellular uptake of siRNA.
We previously reported that LET-mediated cellular uptake of siRNA could be prevented by the macropinocytosis inhibitor amiloride 19 . Macropinocytosis, which is non-selective fluid phase endocytosis, is important for cellular entry of amino acids, virus and electro-transfected plasmid DNA, and is accompanied by actin cytoskeleton remodeling [48][49][50] . We showed that actin cytoskeleton remodeling was induced by LET with siRNA, and this remodeling could be inhibited by pretreatment with EGTA, chelerythrine or 17-DMAG (Fig. 4). Together with our findings that LET-mediated cellular uptake of siRNA was prevented by pretreatment with EGTA or 17-DMAG (Figs 2f and 3f), these results suggested that PKC activation following Ca 2+ influx in the presence of Hsp90α induced actin remodeling, leading to the promotion of cellular uptake of siRNA mediated by LET. Additionally, Rho GTPase regulation of actin remodeling was activated by LET with siRNA, which was also inhibited by pretreatment with EGTA, chelerythrine or 17-DMAG (Fig. 5a). Pretreatment with the Rho inhibitor I prevented not only actin remodeling, but also cellular uptake of siRNA mediated by LET (Fig. 5b,c). Rho GTPase is a subfamily of small GTP-binding proteins of the Ras superfamily, and family members Cdc42, Rac1 and RhoA regulate membrane trafficking through the formation of filopodia, lamellipodia and stress fibers, respectively 40,51 . In the initial phase of macropinocytosis, extensive actin remodeling is required to form the dorsal membrane and peripheral ruffles 40 . Rho GTPase is also involved in formation of ruffles by binding and activating actin nucleators 40 . Therefore, activation of Rho GTPase is vital for the promotion of macropinocytosis and membrane trafficking. Hsp90 was shown to activate Rho GTPase indirectly by protecting functional activators from proteasomal degradation 52 . Moreover, PKCγ activates Rho GTPase thorough phosphorylation of guanine nucleotide exchange factor (GEF) that favors the GTP-bound over GDP-bound form 53 . Taken together, these findings show that Hsp90α and PKCγ promote actin remodeling via the activation of Rho GTPase (Fig. 6).
In conclusion, in this study we demonstrated that LET with siRNA induced the phosphorylation of Hsp90α and PKCγ, which would be both involved in activation of Rho GTPase, indicating that actin remodeling would be important for LET-mediated cellular uptake of siRNA.

Preparation of liposomes.
Liposomes were prepared by a simple lipid film hydration method according to our previous report 22 . Briefly, for preparation of cationic liposomes, EPC and DOTAP dissolved in ethanol were mixed at a molar ratio of 7.6:1, and the ethanol was evaporated under a nitrogen stream to form a thin lipid film. PBS was added to the dried lipid film (total lipid concentration 10 mM) and the suspension was sonicated using a bath-type sonicator (ULTRASONIK 14B, NEY, CA). The anionic liposomes and PEG liposomes were prepared as described above and contained the following lipid compositions: EPC/DCP (9/1 mol/mol) and EPC/Cholesterol/ PEG-DSPE (1.85/1/0.15 mol/mol/mol). Liposome particle sizes and ζ-potentials were examined by dynamic light scattering and laser Doppler, respectively, using a Zetasizer nano (Malvern Ins., Ltd.), and are summarized in Supplementary Table S2  www.nature.com/scientificreports www.nature.com/scientificreports/ iTRAQ, Gene Ontology (GO) slim and STRING analysis. For iTRAQ analysis, NIH 3T3 cells were cultivated at 1.5 × 10 5 cells per dish on 35 mm dishes for 24 h. After washing with PBS (−), cells in PBS (−) were exposed to LET at a constant current of 0.45 mA cm −2 for 15 min. After washing with PBS (−), cells located under the electrode of the cathode were collected and frozen under liquid nitrogen. Untreated cells and cells exposed to LET from three independent experiments were pooled into treated and untreated samples. iTRAQ analysis was carried out at Filgen, Inc. (Nagoya, Japan) according to the manufacturer's protocol accompanying the iTRAQ kit (AB SCIEX, Tokyo, Japan). Briefly, proteins extracted from each of the pooled samples were reductively alkylated and digested with trypsin. The resulting peptide samples were demineralized and labeled with iTRAQ. In this analysis, samples from LET-exposed and untreated cells were labeled with a 116 isobaric tag and 114 isobaric tag, respectively. After concentrating the phosphorylated peptides, identification and relative quantification of phosphorylated proteins were carried out by AB SCIEX Triple TOF TM 5600 System with ProteinPilot TM software 4.1 (AB SCIEX). The thresholds in this study were set as described below. Up-regulated or down-regulated phosphorylated proteins in LET samples were defined as >1.2-fold change relative to untreated cells and a Unused Prot Score >0.62 to show reliabilities of identified proteins. To understand the biological processes, molecular functions and cellular components associated with the phosphorylated proteins that were up-regulated by LET, a GO analysis was carried out at Filgen, Inc. (Nagoya, Japan) using the MicroArray Data Analysis Tool Ver 3.2 (Filgen, Inc). GO slim analysis is a useful tool for understanding the functional role of changed proteins group by statistically addressing the frequency of GO term-attached proteins 54 . To determine interactions among the 15 proteins showing the largest upregulation in phosphorylation following LET, STRING analysis was performed by using STRING ver 10.5 (ELIXIR Core Data Resources).
Western blotting. Western blotting was performed according to our previous report 22 Tween 20). Membranes were incubated with primary antibody (diluted and incubated according to the manufacturer's instructions) followed by incubation with the indicated secondary antibody diluted 1:2000. The blots were detected using ECL Western blotting detection reagent (GE Healthcare, Waukesha, WI) and a C-DiGit blot scanner (LI-COR Biosciences GmbH, Germany). Quantitative analysis of blots was performed by Image Studio Digits software (ver. 5) (LI-COR Biosciences GmbH, Germany). In this analysis, β-actin was used as an internal control. Treatment with pharmacological inhibitors and analysis of cellular uptake of macromolecules. NIH 3T3 cells (1 × 10 5 ) were seeded in 35 mm dishes. After 18 h of cultivation, cells were washed with PBS and then 1 ml serum free DMEM containing either EGTA (5 mM), 17-DMAG (10 μM), chelerythrine (2 μM) or Rho inhibitor I (2 μg/ml) was added into the dish, which was then incubated for 30 min, 1 h, 1 h or 4 h. After incubation, liposomes (final lipid concentration; 25 μM, ratio of rhodamine-PE labelled liposomes to unlabeled liposomes: 1:99) or rhodamine-labelled siRNA (100 pmol) was added to the dishes and the cells were exposed to LET with a constant current of 0.34 mA cm −2 for 15 min. After LET, cells were incubated at 37 °C for 45 min before washing with PBS and lysing with reporter lysis buffer (Promega) according to the manufacturer's protocols. The fluorescence intensity of the cell lysate was measured using a microplate reader Infinite (Tecan Group Ltd., Mannedorf, Switzerland) at excitation and emission wavelengths of 546 nm and 590 nm, respectively.
Evaluation of intracellular Ca 2+ following LET using fluo-4 fluorescent dye. The intracellular amount of Ca 2+ was evaluated using fluo-4 fluorescent dye according to the manufacturer's protocol. Briefly, NIH 3T3 cells (5 × 10 4 cells) were seeded in 35 mm glass bottom dishes coated with 0.002% poly-L-lysine (PLL). After 18 h of cultivation, cells were washed with PBS before 1 ml loading buffer was added to the dish, which was incubated at 37 °C for 1 h. For EGTA treatment, cells were pre-treated with 5 mM EGTA for 30 min. After removing loading buffer, 1 ml pre-warmed recording medium containing liposomes (final lipid concentration: 25 μM) or anti-luciferase siRNA (100 pmol) was added to the dish, followed by LET (0.34 mA/cm 2 ) for 2 min. Immediately after LET, the fluorescence signals corresponding to the amount of intracellular Ca 2+ was observed by Confocal Laser Scanning Microscopy (CLSM) A1R + (Nikon Co., Ltd., Japan).
Actin staining of cells after LET with siRNA. For actin staining, NIH 3T3 cells (5 × 10 4 ) were seeded in 35 mm glass bottom dishes coated with 0.002% PLL. After 18 h of cultivation, cells were washed with PBS and 1 ml serum free DMEM containing liposomes (final lipid concentration 25 μM) or anti-luciferase siRNA (100 pmol) was added before the LET with a constant current of 0.34 mA cm −2 for 15 min. Immediately after LET, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, cells were permeabilized with 1% Triton X-100 at room temperature for 10 min. The cells were then incubated with rhodamine phalloidin at a final concentration of 100 nM for 30 min in dark. After washing, cells were mounted in VECTASHIELD with DAPI and observed by CLSM A1R+ (Nikon Co., Ltd., Japan).