Báo cáo khoa học: Sodium pump a1 and a3 subunit isoforms mediate distinct responses to ouabain and are both essential for survival of human neuroblastoma docx

Sodium pump a1 and a3 subunit isoforms mediate distinct responses to ouabain and are both essential for survival of human neuroblastoma Larisa Karpova 1,2 , Alexander Eva 1 , Ulrike Kirch 1 , Alexander Boldyrev 2 , Georgios Scheiner-Bobis 1 1 Institut fu ¨ r Biochemie und Endokrinologie, Fachbereich Veterina ¨ rmedizin, Justus-Liebig-Universita ¨ t Giessen, Germany 2 Department of Biochemistry, Lomonosov Moscow State University, Moscow, Russia Introduction The sodium pump (Na + ,K + -ATPase; EC 3.6.1.37) maintains the Na + gradient across plasma membranes of animal cells [1]. By hydrolyzing ATP, the enzyme transports three Na + ions out of the cell in exchange for two K + ions that are brought into the cytosol. This activity can be interrupted by a group of substances that are referred to as cardiotonic steroids (CTS), a name linked to their clinical use for the treatment of heart failure [2]. In recent years, numerous publications have established that CTS not only inhibit the sodium pump but also induce signaling cascades that may be associ- ated with cell growth and proliferation as well as with apoptotic cell death, depending upon the cell type or CTS investigated. CTS-induced signaling does not depend on sodium pump inhibition [3–8], as inactive sodium pump mutants can still transmit signals when CTS are added to the cell culture [9,10]. The sodium pump of animal cells is an oligomeric enzyme consisting of a and b subunits [1]. In some tissues, a regulatory c subunit is associated with the a and b subunits [11]. All three subunits have been co-crystallized in several conformational states of the enzyme [12–14]. The a subunit, which is referred to as the catalytic subunit, has ten transmembrane domains, hydrolyzes ATP, transports the cations and is the phar- macological receptor for CTS. The b subunit is a highly glycosylated protein with a single transmembrane span, Keywords cardiotonic steroids; Erk1 ⁄ 2; Na + ,K + -ATPase; signaling; SK-N-AS cells Correspondence G. Scheiner-Bobis, Institut fu ¨ r Biochemie und Endokrinologie, Fachbereich Veterina ¨ rmedizin, Justus-Liebig-Universita ¨ t Giessen, Frankfurter Strasse 100, D-35392 Giessen, Germany Fax: +49 641 9938179 Tel: +49 641 9938180 E-mail: georgios.scheiner-bobis@vetmed. uni-giessen.de (Received 9 December 2009, revised 11 January 2010, accepted 1 February 2010) doi:10.1111/j.1742-4658.2010.07602.x Using SK-N-AS human neuroblastoma cells, which co-express the a1 and a3 isoforms of the sodium pump a subunit, we selectively silenced either the a1ora3 subunit by means of transfection with small interfering RNA, and investigated cell survival and the cellular response to ouabain. We found that both of the a subunits are essential for cell survival, indicating that substitution of one subunit for the other is not sufficient. In the pres- ence of both a subunits, ouabain causes sustained activation of extracellu- lar signal-regulated kinases 1 and 2 (Erk1 ⁄ 2). This activation is not affected when the a1 subunit is silenced. However, when a3 expression is silenced, ouabain-induced activation of Erk1 ⁄ 2 does not occur, even at a high concentration of ouabain (1 lm). Thus, ouabain-induced Erk1 ⁄ 2 acti- vation is mediated in SK-N-AS cells by a3 only, and a1 does not partici- pate in this event. This is a clear demonstration of selective involvement of a specific sodium pump a subunit isoform in ouabain-induced signaling. Abbreviations CTS, cardiotonic steroids; Erk1 ⁄ 2, extracellular signal-regulated kinases 1 and 2; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. FEBS Journal 277 (2010) 1853–1860 ª 2010 The Authors Journal compilation ª 2010 FEBS 1853 and appears to function as a molecular chaperone for correct folding of the a subunit and its transportation to the plasma membrane [15]. The c subunit (also termed FXYD2) is a member of the FXYD family of proteins that includes phospholemman (FXYD1) and corticosteroid hormone-induced factor (FXYD4). These very hydrophobic proteins are characterized by a single transmembrane span (except FXYD3, mammary tumor marker Mat-8, which has two transmembrane spans) and an FYXD motif near the transmembrane domain, in the extracellular N-terminal part of the pro- tein [16]. Each of the subunits exists in various isoforms [1]. There are four a subunits (a1–a4), three b subunits (b1–b3) and two splice variants of the c subunit (FXYD2a and FXYD2b). Various investigations have shown that at least five of the seven FXYD proteins interact with the a and b subunits of Na + ,K + -ATPase and regulate functions of the enzyme [16]. In several cases, multiple isoforms of the a subunit are found in the same cell type, thus raising questions about the physiological significance of such co-existence. Given that CTS ⁄ sodium pump interactions resemble typical hormone ⁄ receptor-mediated events in many respects [7,17], it may be assumed that co-existing subunits are involved in other signaling events in addition to the ion pump function [6–8]; however, there is no direct evidence to support this notion so far. We investigated this question by using the human neuro- blastoma cell line SK-N-AS. These neuroblastoma cells, which co-express the a1 and a3 subunits, were shown to interact with the CTS ouabain. The results provide evidence for distinctive roles in signal media- tion for the two subunit isoforms of the sodium pump. Results Expression of a subunit isoforms of Na + ,K + -ATPase in SK-N-AS cells before and after transfection with Stealth TM RNAi to silence a1 and a3 SK-N-AS is a cancerous neuroblastoma cell line that, like other neuronal cells [18,19], expresses a1 and a3 subunit isoforms (Fig. 1). As shown in Fig. 1A, in control (untreated) SK-N-7AS cells, a1 and a3 subunit- specific cDNA bands are present in equivalent quanti- ties (left lanes). The same result is seen when the cells are transfected with control RNAi: expression of both a1-specific and a3-specific cDNA is like that of the con- trol cells (Fig. 1A, middle lanes). However, transfection Transfected with negative control RNAi M M α2 α3M Control cells Transfected with α α 1 RNAi 500 bp α1 α2 α3 α1 α2 α3 α1 α1 α2 α3 α1 α2 α3 α1 α2 α3 GAPDH H 2 O M Control cells Transfected with negative control RNAi Transfected with α 3 RNAi 500 bp A Control Lipofectamine Negative Control RNAi α 1 isoform of SP RNAi Control Lipofectamine Negative control RNAi α 3 isoform of SP RNAi ** ** 0 25 50 75 100 125 % of control 0 25 50 75 100 125 % of control B Fig. 1. Effect of transfection with various RNAi on expression of a subunit isoforms. (A) Expression of a subunit isoforms in SK-N-AS cells without and with transfection with RNAi. Like other neuronal cells, the neuroblastoma cell line SK-N-AS expresses the a1 and a3 isoforms of the sodium pump a subunit (left lanes, upper and lower panels). Transfection with a1- or a3-specific RNAi silences expression of the corre- sponding mRNA. Detection of glyceraldehyde 3-phosphate dehydrogenase (lower panel, right) was used for normalization purposes. (B) Expression of a subunit isoforms in SK-N-AS cells without and with transfection with RNAi (normalized). Transfection of SK-N-AS cells with either a1- or a3-specific RNAi significantly silences the expression of corresponding mRNA (n =9; **P < 0.01). Lipofectamine alone and negative-control RNAi do not have any effect on the expression of a1ora3 mRNA. Sodium pump isoforms in signaling and survival L. Karpova et al. 1854 FEBS Journal 277 (2010) 1853–1860 ª 2010 The Authors Journal compilation ª 2010 FEBS of the SK-N-AS cells with either a1-specific RNAi (Fig. 1A, upper panel) or a3-specific RNAi (Fig. 1A, lower panel) leads to a reduction in the corresponding mRNA ⁄ cDNA (Fig. 1A, right lanes). In both cases, the reduction in expression of either a1- or a3-specific mRNA is significant (Fig. 1B). mRNA ⁄ cDNA for the a2 subunit was not detected. For normalization of data, glyceraldehyde 3-phosphate dehydrogenase- specific mRNA ⁄ cDNA from every probe was amplified in parallel experiments (Fig. 1A). Its expression was not affected. Survival of SK-N-AS cells before and after transfection with Stealth TM RNAi to silence a1 and a3 Experiments with knockout mice demonstrated that either the a1ora2 isoforms are essential for survival of the animals [20]. Nevertheless, it is not known whether survival of cells that co-express multiple a subunits depends on the simultaneous presence of the various subunits or whether one subunit isoform can substitute for the other. To investigate this question, SK-N-AS cells were treated with specific RNAi to silence the expression of either the a1ora3 subunit of the sodium pump, and cells were cultured for several days. Untreated cells served as a control. The MTT assay was used to determine the number of living cells under each condition. Figure 2 shows that, for the first 48 h, growth is the same for all cell types. However, cells lacking either a1ora3 do not multiply further thereaf- ter, and after 8 days (192 h), the number of living cells was reduced by more than 25% compared to the origi- nal number of cells, and by about 70% when compared to the number of living cells expressing both a1 and a3 subunits. It should be noted, however, that the cells expressing both subunits reached confluence after the 4th day of incubation and therefore did not multiply any further. Ouabain-induced signaling in SK-N-AS cells Ouabain and other CTS induce signaling cascades in a variety of cells and also in the neuroblastoma cell line SH-SY5Y [7,21,22]. One of the first events seen upon exposure of various cell types to CTS is activation of extracellular signal-regulated kinases 1 and 2 (Erk1 ⁄ 2) [3,21,23–26]. Therefore, we focused our attention on 24 48 72 96 120 144 168 192 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Control α1 RNAi α3 RNAi Hours after transfection Absorbance at 540 nm Fig. 2. MTT assay of cell viability. Proliferation of SK-N-AS cells treated with a1- (open circles) or a3- (open triangles) specific RNAi was assessed by the MTT assay over a period of 8 days. Non-trea- ted cells (open squares) served as a control. The proliferation of cells treated with either a1ora3 RNAi declines after 48 h. By the end of the observation time, RNAi-treated cells account for about 30% of the non-treated control (n = 6–12). 50 kDa 40 kDa 30 kDa Phospho Erk 1/2 Ouabain (n M) 0 10 100 1000 0 M M 10 100 1000 Total Erk 1/2 50 kDa 40 kDa 30 kDa 0 10 nM 100 nM 1000 nM 0 100 200 Ouabain Phospho Erk 1/2 kinase (% of control) Phospho Erk 1/2 Ouabain (n M) ** ** ** AB C Fig. 3. Activation of Erk1 ⁄ 2 by ouabain. (A) Detection of total, non-phosphorylated Erk1 ⁄ 2 was used for normalization purposes. (B) Incubation of SK-N-AS cells for 30 min with low concentrations of ouabain stimulates Erk1 ⁄ 2 activation (phosphorylation), as detected by specific antibody against the phosphorylated form of Erk1 ⁄ 2. (C) Erk1 ⁄ 2 activation by ouabain is highly significant at all ouabain concentra- tions tested (n =4;**P < 0.01). M, molecular weight markers. L. Karpova et al. Sodium pump isoforms in signaling and survival FEBS Journal 277 (2010) 1853–1860 ª 2010 The Authors Journal compilation ª 2010 FEBS 1855 possible stimulation (phosphorylation) of these kinases in SK-N-AS cells. As in other cell types, low concentra- tions of ouabain trigger significant activation of Erk1 ⁄ 2 within 30 min in SK-N-AS cells (Fig. 3), thus raising the question of whether ouabain-induced activation of these kinases is mediated through the a1ora3 subunit or through both subunit isoforms of the sodium pump. Ouabain-induced activation of Erk1 ⁄ 2 after silencing either a1ora3 subunits Cells expressing only the ubiquitous a1 subunit of the sodium pump respond to CTS by induction of a variety of signaling cascades. However, in the present study, cells lacking a1 clearly show concentration-dependent ouabain-induced activation of Erk1 ⁄ 2 (Fig. 4). In con- trast, cells lacking a3 did not respond to ouabain, even at the very high concentration of 1 lm (Fig. 4). Discussion Previous experiments with knockout mice have demon- strated that the presence of either a1ora2 subunits is critical for animal survival [20]. However, as single cells can simultaneously express various a subunits, we addressed the question of whether survival of cells would be affected by loss of only one a subunit isoform, and, if so, which one is more essential for survival. Using the same experimental set-up, we also determined whether a1 and a3 subunits respond to ouabain by induction of different signaling events or whether signaling cascades are isoform-independent. When either the a1ora3 subunits are silenced, cells proliferate over a period of 2 days in a manner similar to the control, in which neither of the two subunits had been silenced (Fig. 2). After that, the number of living cells starts declining, until, at day 8, the numbers of cells that lack either a1ora3 are only about 30% of the number of control cells that express both subunit isoforms. The data in Fig. 2 indicate that cells lacking a1 show an earlier loss of viability than those lacking a3; however, the impact of this is not known. Nevertheless, the results clearly show that both a1 and a3 subunits are essential for survival, and that the a1 and a3 isoforms have distinct roles in SK-N-AS cells and loss of one cannot be compensated by the other. Based on these and previous findings, it is possible to speculate that these subunits are similarly essential in other cell types as well. What niche of cell biological functions do a1ora3 subunits occupy that makes them essential for sur- vival? Are their functions identical, or do they differ in some respects? In the investigation presented here, the α3/+ α3/- α3/+ α3/- α3/+ α3/- α3/+ α3/- Ouabain (nM) 0 10 100 1000 0 10 100 1000 α1/+ α1/- α1/+ α1/- α1/+ α1/- α1/+ α1/- Ouabain (nM) 0 50 100 150 200 250 Erk 1/2 activation (%) 0 50 100 150 200 250 Erk 1/2 activation (%) α3/+ α3/- α3/+ α3/- α3/+ α3/- α3/+ α3/- α1/+ α1/- α1/+ α1/- α1/+ α1/- α1/+ α1/- Ouabain (nM)100 100 1000 Ouabain (n M)100 100 1000 * ° * * * * * * * * ° ° ° A B C D Fig. 4. Activation of Erk1 ⁄ 2 by ouabain in cells lacking either a1or a3 subunits. (A) When cells are transfected with a1-specific RNAi (a1 ⁄ -), ouabain still induces activation (phosphorylation) of Erk1 ⁄ 2. (B) When cells are transfected with a3-specific RNAi (a3 ⁄ -), activa- tion of Erk1 ⁄ 2 is absent, even at the high concentration of 1 l M ouabain, indicating involvement of the a3 subunit in the signaling process. (C) Statistical analysis of data similar to those shown in (A) obtained from other western blot experiments, showing stimu- lation of Erk1 ⁄ 2 by ouabain in cells with silenced a1 subunits (asterisk indicates that Erk1 ⁄ 2 is significantly activated at all oua- bain concentrations used in both RNAi-treated and untreated cells; P < 0.05; n = 4). (D) Statistical analysis of data similar to those shown in (B) obtained from other western blot experiments, show- ing stimulation of Erk1 ⁄ 2 by ouabain in cells with silenced a3 subunits (asterisk indicates that Erk1 ⁄ 2 is significantly activated at all ouabain concentrations used in cells not treated with RNAi; P < 0.05; circle indicates significantly reduced Erk1 ⁄ 2 activation in all cells with silenced a3; n = 4). Sodium pump isoforms in signaling and survival L. Karpova et al. 1856 FEBS Journal 277 (2010) 1853–1860 ª 2010 The Authors Journal compilation ª 2010 FEBS latter seems to be the case; cells lacking the a1 subunit respond to ouabain by activation of Erk1 ⁄ 2, indicating that the a3 subunit can transmit CTS-induced signal- ing (Fig. 4A,C). However, silencing the a3 subunit abolishes the ability of the cells to respond to CTS (Fig. 4B), indicating that the presence of a3 is essential for this signaling pathway in SK-N-AS cells. Activa- tion of Erk1 ⁄ 2 was not observed in these cells even at the high concentration of 1 lm ouabain (Fig. 4B), indi- cating that inhibition of the sodium pump, which occurs at this concentration, is not a requirement for CTS-induced signaling, as shown previously [9,10]. Figure 4B,D additionally shows that, after silencing the a3 subunit, the Erk1 ⁄ 2 activity is already reduced compared to the activity seen in control cells, indicat- ing that, even in the absence of externally added oua- bain, part of the Erk1 ⁄ 2 basic activity is contributed by the a3 subunit of the sodium pump. Here we demonstrate for the first time that a1 and a3 have distinct functions in cell physiology, but this leads to a fundamental question: why does a1 not mediate CTS-induced activation of Erk1 ⁄ 2 in SK-N-AS cells when it has been shown to be involved in CTS- induced signaling in various other cells? This question can be addressed by taking into consideration the mechanisms by which CTS generate signals and the structural differences between the a1 and a3 sodium pump subunits. CTS-induced signaling can be explained by two different models. In the first model, it is assumed that cell signaling induced by CTS is due to inhibition of the sodium pump and a local increase in intracellular [Na + ] followed by a subsequent increase in [Ca 2+ ] in the small space between the plasmalemma and endoplasmic ⁄ sarcoplasmic reticulum. This space, referred to as the plasmerosome, contains sodium pump isoforms a 2 and a 3 but not a1 in smooth muscle cells and astrocytes [6,8,27]. In an alternative model, the sodium pump is considered to be a member of a caveolae-defined environment of proteins that are capa- ble of communicating with each other. This entity is referred to as the signalosome [28]. This model pro- poses that it is not inhibition of the sodium pump but rather conformational changes of the CTS ⁄ Na + ,K + -ATPase complex that trigger the signal- ing cascade. This is supported by the fact that signaling cascades are activated when CTS interact with non- pumping sodium pump mutants [9]. Although the models differ in their basic assumption, they have in common the requirement that the sodium pump be targeted to a defined environment. The results presented here show that the a3 isoform can generate CTS-induced activation of Erk1 ⁄ 2, indi- cating its localization in an environment different to that of the a1 isoform. The basis for these different environments is may be due to differences in the struc- ture of the two proteins. The primary structures of the human a1 and a3 isoforms are 87% identical and display a similarity of 94%. Nevertheless, at the level of tertiary structure, they display significant differ- ences. In a recently published comprehensive work, comparison of the tertiary structures of a1 ⁄ a2 and a1 ⁄ a3 subunits revealed that surface-exposed areas of the a2ora3 isoforms were very different from the corresponding areas of the a1 isoform [29]. These areas are found mainly within the N-domain but also within the A-domain of the proteins. The membrane-spanning segments of the three isoforms are rather conserved [29]. We assume that the clusters of isoform-specific differences in the surface-exposed regions might be important for isoform-specific interactions with other proteins. These specific interactions, which may result in either distinctive targeting of the isoforms to differ- ent areas of the plasma membrane or specific interac- tions with signaling molecules (or both), could be the reason for the differences found for ouabain-induced signaling through the a1ora3 isoforms. The recent demonstration that the a1 isoform is recruited to the plasma membrane via interaction of adaptor protein 1 with Tyr255 of this isoform supports this hypothesis, and demonstrates that even small differences in surface-exposed areas may have a big impact in target- ing of the proteins [30]. Isoforms a2ora3 lack this tyrosine residue, and we assume that their targeting to specific areas of the plasma membrane must be defined by other parameters. Thus, based on our results and those of others discussed above, we suggest that the differences seen in the CTS-induced signaling through the a1ora3 isoforms are associated with differences in the surface- exposed regions of the two proteins. These might lead to specific targeting of each isoform to different micro-environments of the plasma membrane or to isoform-specific interactions with other proteins of the micro-environments. Future work should help to verify this assumption. Experimental procedures Cell culture SK-N-AS cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (PromoCell, Heidelberg, Germany) supple- mented with 10% v ⁄ v fetal bovine serum (PromoCell) and 100 IUÆmL- 1 Pen ⁄ Strep (PromoCell). The culture was main- tained in a humidified incubator at 37 °Cin5%CO 2 . The L. Karpova et al. Sodium pump isoforms in signaling and survival FEBS Journal 277 (2010) 1853–1860 ª 2010 The Authors Journal compilation ª 2010 FEBS 1857 medium was replaced twice per week. Cells were harvested by incubating with trypsin (0.25%; PromoCell) for 2 min at 37 °C. Preparation of cell lysates Cell lysates were prepared as described previously [21]. SDS/PAGE and western blotting of isolated proteins A total of 10–50 lg of protein was separated by SDS/PAGE using 10% acrylamide and 0.3% N,N¢-methylene-bis-acryla- mide gels. Biotinylated molecular weight markers (Cell Signaling Technology, Frankfurt am Main, Germany) were run in parallel. After SDS/PAGE, proteins were electro- blotted onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) at 500 mA for 30–40 min. Detection of proteins was performed as described by the manufacturers of the antibodies (Cell Signaling Technology, Santa Cruz Inc. or Dianova, Hamburg, Germany) in combination with an enhanced chemiluminescence (ECL) kit (GE HealthCare, Munich, Germany). Chemiluminescence was visualized and quantified using a molecular imager ChemiDoc XRS system (Bio-Rad, Munich, Germany). Detection of a1-, a2- and a3-specific mRNA in SK-N-AS cells Cells were grown to 75% confluence before isolation of total mRNA using the RNeasy mini kit (Qiagen, Hilden, Germany). RNase-free DNase I (Qiagen) was used to elimi- nate potential contamination by DNA. The concentration and purity of total mRNA were determined by measuring the absorbance at 260 and 280 nm. The OneStep RT-PCR kit (Qiagen) was used for reverse transcription and PCR amplification of DNA. In a total vol- ume of 50 lL, 20–40 ng of mRNA, 1 lL (20 pmolÆmL )1 )of each primer, 10 lLof5· buffer (12.5 mmolÆL )1 MgCl 2 , 20 mmolÆL )1 Tris ⁄ HCl, 100 mmolÆL )1 KCl, 10 mmolÆL )1 dNTPs), 2 lL of a mixture of Omniscript and Sensiscript reverse transcriptases and HotStar Taq DNA polymerase were incubated in a MasterCycler gradient (Eppendorf, Hamburg, Germany) at 50 °C for 30 min for the reverse transcription reaction. Then the mixture was heated at 95 ° C for 15 min, followed by 40–45 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min. For specific amplification of a1, the forward and reverse primers were 5¢-GTTGGGGCTCCGATGTGTT GGGGT-3¢ and 5¢-CTGGCTGGAGGCTGTCATCTTCTT CAT-3¢, respectively; for specific amplification of a2, the forward and reverse primers were 5¢-CTGGCTGGAGGC TGTCATCTTCTTCAT-3¢ and 5¢-GGCTCTTGGGGGCT GTCTTCTCGCT-3¢, respectively; for specific amplification of a3, the forward and reverse primers were 5¢-CTGGCTT GAGGCTGTCATCTTCTTCAT-3¢ and 5¢-ATCGGTTGT CGTTGGGGTCCTCGGT-3¢ respectively. The correspond- ing fragment sizes are 560 bp (a1), 557 bp (a2) and 560 bp (a3). To control PCR efficiency and the quality of the cDNA, primers 5¢-TGGGGAAGGTGAAGGTCGGAGTCAA-3¢ and 5¢-TAAGCAGTTGGTGGTGCAGGAGGCA-3¢ were used to co-amplify a specific fragment of 469 bp coding for the housekeeping gene glyceraldehyde 3-phosphate dehydro- genase. The RT-PCR products were analyzed by electrophoresis in a 1.7% agarose gel. The correct identity of a1- or a3-spe- cific amplified sequences was further verified by digestion with Bpi, Eco47I, BspTI (all MBI Fermentas, St Leon-Rot, Germany) and subsequent agarose gel electrophoresis. Silencing a1 or a3 mRNA biosynthesis by siRNA transfection Stealth TM RNAi (Invitrogen, Karlsruhe, Germany) for silencing a1 and a3 isoforms of Na + ,K + -ATPase was transfected into SK-N-AS cells using Lipofectamine 2000 according to the manufacturer’s protocol. In brief, 2 · 10 5 SK-N-AS cells were placed in each well of a six-well culture vessel in Dulbecco’s modified Eagle’s medium without antibiotics. Cells were 30–50% confluent at the time of transfection. Before transfection, Stealth TM RNAi and Lipo- fectamine 2000 were diluted with Opti MEM I Reduced Serum Medium (Invitrogen) and incubated for 5 min at room temperature. Then, diluted Stealth TM RNAi and diluted Lipofectamine 2000 were combined, mixed gently and added to the cells after 20 min. The final concentration of the Stealth TM RNAi was 100 nm. Control cells were trea- ted with Lipofectamine 2000 only. In parallel, cells were transfected with Stealth TM RNAi Negative Control High or Medium GC (negative control for either a1ora3Na + ,K + - ATPase Stealth TM RNAi). After 72 h of incubation at 37 °C in a CO 2 incubator, transfection efficiencies of 81 ± 3% in the SK-N-AS cells were estimated using a Block-iT TM trans- fection kit (Invitrogen) according the manufacturer’s proto- col. Total RNA was isolated from SK-N-AS cells, and the extracted RNA was subjected to RT-PCR to amplify a1-, a2- and a3-specific DNA fragments as described above. The sequences for the Stealth TM RNAi were: 5¢-GGG UGUGGUGCUAUCAGCCGUUGUA-3¢ and 5¢-UACAA CGGCUGAUAGCACCACACCC-3¢ (a1 subunit); 5¢-AC GACAACCGAUACCUGCUGGUGAU-3¢ and ‘5-AUCA CCAGCAGGUAUCGGUUGUCGU-3¢ (a3 subunit). Seventy-two hours after transfection with Stealth TM RNAi, cells were exposed to various ouabain concentrations for 3 h. These cells were used to produce lysates that were subsequently used in western blot experiments. In a different set of experiments, the survival of cells transfected with Stealth TM RNAi was followed over a longer period of time using the MTT assay described below. Sodium pump isoforms in signaling and survival L. Karpova et al. 1858 FEBS Journal 277 (2010) 1853–1860 ª 2010 The Authors Journal compilation ª 2010 FEBS MTT assay Cells were treated with Stealth TM RNAi (2 · 10 4 cells per well ⁄ 24-well culture vessel) under the conditions described above. After replacing the transfection medium by complete growth medium, incubation was continued for various times (1–8 days). Afterwards, the medium was aspirated and replaced by 300 lL of fresh medium containing 0.5 mgÆmL )1 MTT. The cells were incubated for an addi- tional 4 h. 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Sodium pump a1 and a3 subunit isoforms mediate distinct responses to ouabain and are both essential for survival of human neuroblastoma Larisa Karpova 1,2 , Alexander Eva 1 , Ulrike. the a1ora3 subunit or through both subunit isoforms of the sodium pump. Ouabain- induced activation of Erk1 ⁄ 2 after silencing either a1ora3 subunits Cells expressing only the ubiquitous a1 subunit. those lacking a3; however, the impact of this is not known. Nevertheless, the results clearly show that both a1 and a3 subunits are essential for survival, and that the a1 and a3 isoforms have distinct