- Email: [email protected]
A limitation of the method for siRNA delivery into primary human cytotrophoblast cells
Placenta 32 (2011) 192e194
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Technical Note
A limitation of the method for siRNA delivery into primary human cytotrophoblast cells M. Desforges*, M. Westwood Maternal and Fetal Health Research Centre, Developmental Biomedicine, School of Biomedicine, Manchester Academic Health Sciences Centre, University of Manchester, St Mary’s Hospital, Oxford Road, Manchester M13 9WL, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 15 November 2010
Transfection using cationic lipid reagents such as DharmaFECT2 is an efficient tool for introducing siRNA into primary human cytotrophoblast cells. However, we now report a limitation to the use of this method as the concentration of DharamFECT2 needed for effective siRNA delivery causes ligand-independent activation of the insulin/insulin-like growth factor (IGF) receptor and consequently, functional resistance to cytotrophoblast stimulation by these two hormones. We therefore advise researchers against the use of this method of transfection when investigating the mechanisms by which insulin/IGF, and potentially other hormones that exert their effects through kinase signalling molecules, influence cytotrophoblast cell function. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: DharmaFECT Transfection Insulin IGF-I Insulin receptor
1. Introduction We recently tested a variety of technological approaches and reagents for introducing siRNA into primary cytotrophoblast cells isolated from term human placenta and, based on efficiency of siRNA delivery, degree of target knockdown and cell viability, reported transfection using the lipid-based transfection reagent, DharmaFECT2 as the optimal method [1]. This protocol has been used by our Centre and other laboratories as a tool for examining the function(s) of a variety of proteins expressed in human placenta [2e6]. Indeed this approach revealed that the SNAT1 subtype of the system A amino acid transporter makes a substantial contribution to the total activity of this system in term placenta [2], and so we next sought to investigate if stimulation of system A activity by insulin and insulin-like growth factor I (IGF-I) [7e9] is a SNAT1mediated response. Our data from untransfected cells confirm that system A is regulated by these two hormones. However, we were unable determine the involvement of SNAT1 because exposure to the transfection reagent rendered cytotrophoblast cells unresponsive to insulin/IGF-I stimulation.
* Corresponding author. Maternal and Fetal Health Research Centre, Developmental Biomedicine, University of Manchester, St Mary’s Hospital, Level 5-Research, Manchester M13 9WL, United Kingdom. Tel.: þ44 (0) 161 7016962; fax: þ44 (0) 161 7016971. E-mail address: [email protected] (M. Desforges). 0143-4004/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.11.013
2. Methods 2.1. Cytotrophoblast cell culture and system A activity measurements Term placentas were collected from uncomplicated singleton pregnancies with written informed consent and in accordance with Local Ethics Committee approval. Cytotrophoblast cells were isolated, plated onto P35 dishes (Nunc, Denmark) at a density of 2e2.5 106, and maintained in primary culture [10]. At 18 h of culture, cytotrophoblast cells were transfected with SNAT1-specific siRNA (Qiagen) using a protocol previously developed to achieve maximum SNAT1 knockdown in conjunction with minimum exposure to reagents (50 nM siRNA, 3 ml DharmaFECT2 transfection reagent (Dharmacon) in a total volume of 1.5 ml [1,2]). Untransfected cells, cells transfected with non-targeting siRNA (Invitrogen), and cells exposed to only DharmaFECT2 (i.e. mock-transfected) were included as controls. At 62 h of culture, control and transfected cells were cultured with and without insulin (300 ng/ ml) or IGF-I (100 ng/ml or 250 ng/ml), then 4 h later, uptake of the radiolabelled system A-specific substrate, 14C-MeAIB (10 nM), was measured over 20 min [2].
2.2. Western blot analysis of insulin/IGF-I receptor activation Cytotrophoblast cell lysates were analysed for insulin receptor/insulin-like growth factor-1 receptor (IR/IGF1R) b subunit expression and phosphorylation by Western blotting using established methods [11] with the following modifications. Nitrocellulose membranes were blocked for 2 h (5% BSA in TBS-0.1% Tween 20) then incubated at 4 C overnight with an antibody specific to phosphorylated IR/IGF1Rb (1:1000 dilution in blocking buffer; Invitrogen clone pYpY1162/1163). Membranes were washed with TBS-0.1% Tween 20 then incubated for 2 h at room temperature with species-specific HRP-conjugated secondary antibody (Dako). Immunoreactive proteins were visualized using enhanced chemiluminescence. The membrane was stripped [12] and reprobed with an antibody specific for total IGF1Rb (1:1000; Santa Cruz Biotechnology clone c-20).
M. Desforges, M. Westwood / Placenta 32 (2011) 192e194
193
Fig. 1. The effect of SNAT1 knockdown on 14C-MeAIB uptake by cytotrophoblast cells treated for 4 h with; a) 300 ng/ml insulin, and b) IGF-I. Data from each individual experiment/ cytotrophoblast cell preparation are shown as aligned squares. Closed squares ¼ untransfected control cells, open squares ¼ cells transfected with 50 nM SNAT1-specific siRNA.
3. Results & discussion 3.1. Effect of insulin and IGF-I on system A activity in cytotrophoblast cells Basal 14C-MeAIB uptake by untransfected cytotrophoblast cells ranged from 97.24 to 497.04 pmol/mg protein/20 min (n ¼ 6). In agreement with the accumulating evidence for the regulation of system A by insulin and IGF-I [7e9], exposure of untransfected cytotrophoblast cells to these hormones increased 14C-MeAIB uptake by 20e68% (n ¼ 6, p < 0.05 wilcoxon signed rank) and
30e78% (n ¼ 4) respectively. We next investigated the role of SNAT1 in this process by using siRNA to reduce SNAT1 expression. Neither mock-transfection nor transfection with non-targeting siRNA affected basal 14C-MeAIB uptake by cytotrophoblast cells but uptake was significantly reduced by 41e75% following transfection with SNAT1-specific siRNA (n ¼ 5, p < 0.05 wilcoxon signed rank), thereby confirming our assertion that this isoform makes a significant contribution to total system A activity in cytotrophoblast [2]. Interestingly, in each experiment performed, the ability of insulin and IGF-I to stimulate 14C-MeAIB uptake by cells with reduced SNAT1 was attenuated (Fig. 1a and b), suggesting these hormones
Fig. 2. a) The effect of transfection on 14C-MeAIB uptake by cytotrophoblast cells treated for 4 h with 300 ng/ml insulin or 100 ng/ml IGF-I. Closed squares ¼ untransfected control cells, open squares ¼ cells transfected with 50 nM SNAT1-specific siRNA, triangles ¼ cells transfected with 50 nM non-targeting siRNA, circles ¼ mock-transfected cells. b) Western blot analysis of phosphorylated and total IR/IGF1R in cytotrophoblast cell lysates. Cells were transfected at 18 h of culture and then either harvested after 4 h (22 h cells) or maintained in culture and treated with insulin (300 ng/ml) for 4 h before harvesting at 66 h. Immunoreactive signals were observed at the expected size of w95 kDa. Absence of immunoreactive signal in parallel blots probed with pYpY1162/1163 primary antibody preabsorbed with an excess of antigenic phospho-peptide (10) confirmed specificity of the signals. 100 mg protein loaded per sample. C ¼ untransfected control cells, M ¼ mock-transfected cells, N50/100 ¼ cells transfected with 50/100 nM non-targeting siRNA, þI ¼ exposure to insulin.
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regulate system A-mediated amino acid uptake by influencing SNAT1 activity. However, upregulation of system A activity by insulin and IGF-I was also prevented in cells that had been mocktransfected or transfected with non-targeting siRNA and therefore had intact SNAT1 activity (Fig. 2a). This led us to question if the transfection procedure had rendered the cells unresponsive to these hormones; studies of other cell types have shown that lipidbased transfection reagents can cause activation of insulin receptor kinases independently of hormone stimulation [13] and we therefore investigated insulin receptor/IGF-I receptor phosphorylation status in untransfected and transfected cytotrophoblast cells.
3.2. Activation of insulin/IGF-I receptors following transfection of cytotrophoblast cells Western blot analysis of mock-transfected cells demonstrated that even a 4 h exposure to DharmaFECT2 is sufficient to activate IR/IGF1Rb (Fig. 2b, 22 h cells). DharmaFECT is a cationic lipid and therefore has the potential to alter the physical properties of the cell membrane; this in turn could cause autophosphorylation of these receptors [14,15]. However, the effect of charge is unlikely to be the sole explanation of our findings as a similar level of IR/IGF1Rb activation was observed 4 h after transfection with the (ostensibly) neutral complex formed by mixing the transfection reagent with 50 nM or 100 nM non-targeting siRNA (Fig. 2b, 22 h cells). The activation of IR/IGF1Rb in mock-transfected cells at 48 h posttransfection (i.e. 66 h of cultureethe timepoint at which amino acid uptake was assessed), was more pronounced and of a similar level to that caused by insulin stimulation of untransfected cells (Fig. 2b). Indeed, insulin treatment of mock-transfected cells had no additional affect on IR/IGF1Rb phosphorylation status, which supports the hypothesis that DharmeFECT2 causes cellular insensitivity to insulin/IGF-I by maximally activating IR/IGF1Rb.
3.3. Summary and conclusion Here we describe a limitation of the method for transfection of primary human cytotrophoblast cells with siRNA; DharmaFECT2, like other lipid-based transfection reagents [13], causes insulin/ IGF-I receptor autophosphorylation and consequently, resistance to further activation by their cognate ligands. Such reagents also cause non-specific activation of other intracellular signalling molecules, e.g. Stat3, Akt and mTOR [16,17]. Although judicious use of controls will enable accurate interpretation of data, researchers are advised that this experimental protocol, may not be suitable for investigating the mechanisms by which insulin/IGF, and potentially other hormones/growth factors dependent on kinase signalling pathways, regulate cytotrophoblast cell function.
Acknowledgements The authors thank the midwives of St. Mary’s Hospital for their assistance in obtaining placentas. This work has been funded by The Wellcome Trust (078814/Z/ 05/Z) and we acknowledge core support from the Manchester NIHR Biomedical Research Centre and the NIHR Greater Manchester Comprehensive Local Research Network. References [1] Forbes K, Desforges M, Garside R, Aplin JD, Westwood M. Methods for siRNAmediated reduction of mRNA and protein expression in human placental explants, isolated primary cells and cell lines. Placenta 2009 Feb;30(2):124e9. [2] Desforges M, Greenwood SL, Glazier JD, Westwood M, Sibley CP. The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast. Biochem Biophys Res Commun 2010;398:130e4. [3] Desforges M, Sibley CP, Westwood M, Greenwood SL. Reduced system beta transporter activity in primary cytotrophoblast cells increases susceptibility to apoptotic stimuli. Reprod Sci 2010;17(3):317A. [4] Jones HN, Jansson T, Powell TL. IL-6 stimulates system A amino acid transporter activity in trophoblast cells through STAT3 and increased expression of SNAT2. Am J Physiol Cell Physiol 2009 Nov;297(5):C1228e35. [5] Parsons L, Greenwood SL, Westwood M, Desforges M. The effect of reduced system beta transporter activity on cytotrophoblast cell Differentiation in Vitro. Placenta 2009;30:A 83. [6] Rosario FJ, Powell TL, Jansson T. mTORC1 and mTORC2 regukates placental amino acid transporters. Reprod Sci 2010;17(3):259A. [7] Karl P. Insulin-like growth factor-I stimulates amino acid uptake by the cultured human placental trophoblast. J Cell Physiol 1995;165:83e8. [8] Karl PI, Alpy KL, Fisher SE. Amino acid transport by the cultured human placental trophoblast: effect of insulin on AIB transport. Am J Physiol 1992 Apr;262(4 Pt 1):C834e9. [9] Roos S, Lagerlof O, Wennergren M, Powell TL, Jansson T. Regulation of amino acid transporters by glucose and growth factors in cultured primary human trophoblast cells is mediated by mTOR signaling. Am J Physiol Cell Physiol 2009 Sep;297(3):C723e31. [10] Greenwood SL, Brown PD, Edwards D, Sibley CP. Patch clamp studies of human placental cytotrophoblast cells in culture. Troph Res 1993;7:53e68. [11] Matthews LC, Taggart MJ, Westwood M. Effect of cholesterol depletion on mitogenesis and survival: the role of caveolar and noncaveolar domains in insulin-like growth factor-mediated cellular function. Endocrinology 2005 Dec;146(12):5463e73. [12] Forbes K, West G, Garside R, Aplin JD, Westwood M. The protein-tyrosine phosphatase, SRC homology-2 domain containing protein tyrosine phosphatase-2, is a crucial mediator of exogenous insulin-like growth factor signaling to human trophoblast. Endocrinology 2009 Oct;150(10):4744e54. [13] Pramfalk C, Lanner J, Andersson M, Danielsson E, Kaiser C, Renstrom IM, et al. Insulin receptor activation and down-regulation by cationic lipid transfection reagents. BMC Cell Biol 2004 Jan 26;5:7. [14] Koltover I, Salditt T, Radler JO, Safinya CR. An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery. Science 1998 Jul 3;281(5373):78e81. [15] McCallum CD, Epand RM. Insulin receptor autophosphorylation and signaling is altered by modulation of membrane physical properties. Biochemistry 1995 Feb 14;34(6):1815e24. [16] Arulanandam R, Vultur A, Raptis L. Transfection techniques affecting Stat3 activity levels. Anal Biochem 2005 Mar 1;338(1):83e9. [17] Verreault M, Bally MB. siRNA-mediated integrin-linked kinase suppression: nonspecific effects of siRNA/cationic liposome complexes trigger changes in the expression of phosphorylated-AKT and mTOR independently of ILK silencing. Oligonucleotides 2009 Jun;19(2):129e40.
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Technical Note
A limitation of the method for siRNA delivery into primary human cytotrophoblast cells M. Desforges*, M. Westwood Maternal and Fetal Health Research Centre, Developmental Biomedicine, School of Biomedicine, Manchester Academic Health Sciences Centre, University of Manchester, St Mary’s Hospital, Oxford Road, Manchester M13 9WL, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 15 November 2010
Transfection using cationic lipid reagents such as DharmaFECT2 is an efficient tool for introducing siRNA into primary human cytotrophoblast cells. However, we now report a limitation to the use of this method as the concentration of DharamFECT2 needed for effective siRNA delivery causes ligand-independent activation of the insulin/insulin-like growth factor (IGF) receptor and consequently, functional resistance to cytotrophoblast stimulation by these two hormones. We therefore advise researchers against the use of this method of transfection when investigating the mechanisms by which insulin/IGF, and potentially other hormones that exert their effects through kinase signalling molecules, influence cytotrophoblast cell function. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: DharmaFECT Transfection Insulin IGF-I Insulin receptor
1. Introduction We recently tested a variety of technological approaches and reagents for introducing siRNA into primary cytotrophoblast cells isolated from term human placenta and, based on efficiency of siRNA delivery, degree of target knockdown and cell viability, reported transfection using the lipid-based transfection reagent, DharmaFECT2 as the optimal method [1]. This protocol has been used by our Centre and other laboratories as a tool for examining the function(s) of a variety of proteins expressed in human placenta [2e6]. Indeed this approach revealed that the SNAT1 subtype of the system A amino acid transporter makes a substantial contribution to the total activity of this system in term placenta [2], and so we next sought to investigate if stimulation of system A activity by insulin and insulin-like growth factor I (IGF-I) [7e9] is a SNAT1mediated response. Our data from untransfected cells confirm that system A is regulated by these two hormones. However, we were unable determine the involvement of SNAT1 because exposure to the transfection reagent rendered cytotrophoblast cells unresponsive to insulin/IGF-I stimulation.
* Corresponding author. Maternal and Fetal Health Research Centre, Developmental Biomedicine, University of Manchester, St Mary’s Hospital, Level 5-Research, Manchester M13 9WL, United Kingdom. Tel.: þ44 (0) 161 7016962; fax: þ44 (0) 161 7016971. E-mail address: [email protected] (M. Desforges). 0143-4004/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.11.013
2. Methods 2.1. Cytotrophoblast cell culture and system A activity measurements Term placentas were collected from uncomplicated singleton pregnancies with written informed consent and in accordance with Local Ethics Committee approval. Cytotrophoblast cells were isolated, plated onto P35 dishes (Nunc, Denmark) at a density of 2e2.5 106, and maintained in primary culture [10]. At 18 h of culture, cytotrophoblast cells were transfected with SNAT1-specific siRNA (Qiagen) using a protocol previously developed to achieve maximum SNAT1 knockdown in conjunction with minimum exposure to reagents (50 nM siRNA, 3 ml DharmaFECT2 transfection reagent (Dharmacon) in a total volume of 1.5 ml [1,2]). Untransfected cells, cells transfected with non-targeting siRNA (Invitrogen), and cells exposed to only DharmaFECT2 (i.e. mock-transfected) were included as controls. At 62 h of culture, control and transfected cells were cultured with and without insulin (300 ng/ ml) or IGF-I (100 ng/ml or 250 ng/ml), then 4 h later, uptake of the radiolabelled system A-specific substrate, 14C-MeAIB (10 nM), was measured over 20 min [2].
2.2. Western blot analysis of insulin/IGF-I receptor activation Cytotrophoblast cell lysates were analysed for insulin receptor/insulin-like growth factor-1 receptor (IR/IGF1R) b subunit expression and phosphorylation by Western blotting using established methods [11] with the following modifications. Nitrocellulose membranes were blocked for 2 h (5% BSA in TBS-0.1% Tween 20) then incubated at 4 C overnight with an antibody specific to phosphorylated IR/IGF1Rb (1:1000 dilution in blocking buffer; Invitrogen clone pYpY1162/1163). Membranes were washed with TBS-0.1% Tween 20 then incubated for 2 h at room temperature with species-specific HRP-conjugated secondary antibody (Dako). Immunoreactive proteins were visualized using enhanced chemiluminescence. The membrane was stripped [12] and reprobed with an antibody specific for total IGF1Rb (1:1000; Santa Cruz Biotechnology clone c-20).
M. Desforges, M. Westwood / Placenta 32 (2011) 192e194
193
Fig. 1. The effect of SNAT1 knockdown on 14C-MeAIB uptake by cytotrophoblast cells treated for 4 h with; a) 300 ng/ml insulin, and b) IGF-I. Data from each individual experiment/ cytotrophoblast cell preparation are shown as aligned squares. Closed squares ¼ untransfected control cells, open squares ¼ cells transfected with 50 nM SNAT1-specific siRNA.
3. Results & discussion 3.1. Effect of insulin and IGF-I on system A activity in cytotrophoblast cells Basal 14C-MeAIB uptake by untransfected cytotrophoblast cells ranged from 97.24 to 497.04 pmol/mg protein/20 min (n ¼ 6). In agreement with the accumulating evidence for the regulation of system A by insulin and IGF-I [7e9], exposure of untransfected cytotrophoblast cells to these hormones increased 14C-MeAIB uptake by 20e68% (n ¼ 6, p < 0.05 wilcoxon signed rank) and
30e78% (n ¼ 4) respectively. We next investigated the role of SNAT1 in this process by using siRNA to reduce SNAT1 expression. Neither mock-transfection nor transfection with non-targeting siRNA affected basal 14C-MeAIB uptake by cytotrophoblast cells but uptake was significantly reduced by 41e75% following transfection with SNAT1-specific siRNA (n ¼ 5, p < 0.05 wilcoxon signed rank), thereby confirming our assertion that this isoform makes a significant contribution to total system A activity in cytotrophoblast [2]. Interestingly, in each experiment performed, the ability of insulin and IGF-I to stimulate 14C-MeAIB uptake by cells with reduced SNAT1 was attenuated (Fig. 1a and b), suggesting these hormones
Fig. 2. a) The effect of transfection on 14C-MeAIB uptake by cytotrophoblast cells treated for 4 h with 300 ng/ml insulin or 100 ng/ml IGF-I. Closed squares ¼ untransfected control cells, open squares ¼ cells transfected with 50 nM SNAT1-specific siRNA, triangles ¼ cells transfected with 50 nM non-targeting siRNA, circles ¼ mock-transfected cells. b) Western blot analysis of phosphorylated and total IR/IGF1R in cytotrophoblast cell lysates. Cells were transfected at 18 h of culture and then either harvested after 4 h (22 h cells) or maintained in culture and treated with insulin (300 ng/ml) for 4 h before harvesting at 66 h. Immunoreactive signals were observed at the expected size of w95 kDa. Absence of immunoreactive signal in parallel blots probed with pYpY1162/1163 primary antibody preabsorbed with an excess of antigenic phospho-peptide (10) confirmed specificity of the signals. 100 mg protein loaded per sample. C ¼ untransfected control cells, M ¼ mock-transfected cells, N50/100 ¼ cells transfected with 50/100 nM non-targeting siRNA, þI ¼ exposure to insulin.
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M. Desforges, M. Westwood / Placenta 32 (2011) 192e194
regulate system A-mediated amino acid uptake by influencing SNAT1 activity. However, upregulation of system A activity by insulin and IGF-I was also prevented in cells that had been mocktransfected or transfected with non-targeting siRNA and therefore had intact SNAT1 activity (Fig. 2a). This led us to question if the transfection procedure had rendered the cells unresponsive to these hormones; studies of other cell types have shown that lipidbased transfection reagents can cause activation of insulin receptor kinases independently of hormone stimulation [13] and we therefore investigated insulin receptor/IGF-I receptor phosphorylation status in untransfected and transfected cytotrophoblast cells.
3.2. Activation of insulin/IGF-I receptors following transfection of cytotrophoblast cells Western blot analysis of mock-transfected cells demonstrated that even a 4 h exposure to DharmaFECT2 is sufficient to activate IR/IGF1Rb (Fig. 2b, 22 h cells). DharmaFECT is a cationic lipid and therefore has the potential to alter the physical properties of the cell membrane; this in turn could cause autophosphorylation of these receptors [14,15]. However, the effect of charge is unlikely to be the sole explanation of our findings as a similar level of IR/IGF1Rb activation was observed 4 h after transfection with the (ostensibly) neutral complex formed by mixing the transfection reagent with 50 nM or 100 nM non-targeting siRNA (Fig. 2b, 22 h cells). The activation of IR/IGF1Rb in mock-transfected cells at 48 h posttransfection (i.e. 66 h of cultureethe timepoint at which amino acid uptake was assessed), was more pronounced and of a similar level to that caused by insulin stimulation of untransfected cells (Fig. 2b). Indeed, insulin treatment of mock-transfected cells had no additional affect on IR/IGF1Rb phosphorylation status, which supports the hypothesis that DharmeFECT2 causes cellular insensitivity to insulin/IGF-I by maximally activating IR/IGF1Rb.
3.3. Summary and conclusion Here we describe a limitation of the method for transfection of primary human cytotrophoblast cells with siRNA; DharmaFECT2, like other lipid-based transfection reagents [13], causes insulin/ IGF-I receptor autophosphorylation and consequently, resistance to further activation by their cognate ligands. Such reagents also cause non-specific activation of other intracellular signalling molecules, e.g. Stat3, Akt and mTOR [16,17]. Although judicious use of controls will enable accurate interpretation of data, researchers are advised that this experimental protocol, may not be suitable for investigating the mechanisms by which insulin/IGF, and potentially other hormones/growth factors dependent on kinase signalling pathways, regulate cytotrophoblast cell function.
Acknowledgements The authors thank the midwives of St. Mary’s Hospital for their assistance in obtaining placentas. This work has been funded by The Wellcome Trust (078814/Z/ 05/Z) and we acknowledge core support from the Manchester NIHR Biomedical Research Centre and the NIHR Greater Manchester Comprehensive Local Research Network. References [1] Forbes K, Desforges M, Garside R, Aplin JD, Westwood M. Methods for siRNAmediated reduction of mRNA and protein expression in human placental explants, isolated primary cells and cell lines. Placenta 2009 Feb;30(2):124e9. [2] Desforges M, Greenwood SL, Glazier JD, Westwood M, Sibley CP. The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast. Biochem Biophys Res Commun 2010;398:130e4. [3] Desforges M, Sibley CP, Westwood M, Greenwood SL. Reduced system beta transporter activity in primary cytotrophoblast cells increases susceptibility to apoptotic stimuli. Reprod Sci 2010;17(3):317A. [4] Jones HN, Jansson T, Powell TL. IL-6 stimulates system A amino acid transporter activity in trophoblast cells through STAT3 and increased expression of SNAT2. Am J Physiol Cell Physiol 2009 Nov;297(5):C1228e35. [5] Parsons L, Greenwood SL, Westwood M, Desforges M. The effect of reduced system beta transporter activity on cytotrophoblast cell Differentiation in Vitro. Placenta 2009;30:A 83. [6] Rosario FJ, Powell TL, Jansson T. mTORC1 and mTORC2 regukates placental amino acid transporters. Reprod Sci 2010;17(3):259A. [7] Karl P. Insulin-like growth factor-I stimulates amino acid uptake by the cultured human placental trophoblast. J Cell Physiol 1995;165:83e8. [8] Karl PI, Alpy KL, Fisher SE. Amino acid transport by the cultured human placental trophoblast: effect of insulin on AIB transport. Am J Physiol 1992 Apr;262(4 Pt 1):C834e9. [9] Roos S, Lagerlof O, Wennergren M, Powell TL, Jansson T. Regulation of amino acid transporters by glucose and growth factors in cultured primary human trophoblast cells is mediated by mTOR signaling. Am J Physiol Cell Physiol 2009 Sep;297(3):C723e31. [10] Greenwood SL, Brown PD, Edwards D, Sibley CP. Patch clamp studies of human placental cytotrophoblast cells in culture. Troph Res 1993;7:53e68. [11] Matthews LC, Taggart MJ, Westwood M. Effect of cholesterol depletion on mitogenesis and survival: the role of caveolar and noncaveolar domains in insulin-like growth factor-mediated cellular function. Endocrinology 2005 Dec;146(12):5463e73. [12] Forbes K, West G, Garside R, Aplin JD, Westwood M. The protein-tyrosine phosphatase, SRC homology-2 domain containing protein tyrosine phosphatase-2, is a crucial mediator of exogenous insulin-like growth factor signaling to human trophoblast. Endocrinology 2009 Oct;150(10):4744e54. [13] Pramfalk C, Lanner J, Andersson M, Danielsson E, Kaiser C, Renstrom IM, et al. Insulin receptor activation and down-regulation by cationic lipid transfection reagents. BMC Cell Biol 2004 Jan 26;5:7. [14] Koltover I, Salditt T, Radler JO, Safinya CR. An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery. Science 1998 Jul 3;281(5373):78e81. [15] McCallum CD, Epand RM. Insulin receptor autophosphorylation and signaling is altered by modulation of membrane physical properties. Biochemistry 1995 Feb 14;34(6):1815e24. [16] Arulanandam R, Vultur A, Raptis L. Transfection techniques affecting Stat3 activity levels. Anal Biochem 2005 Mar 1;338(1):83e9. [17] Verreault M, Bally MB. siRNA-mediated integrin-linked kinase suppression: nonspecific effects of siRNA/cationic liposome complexes trigger changes in the expression of phosphorylated-AKT and mTOR independently of ILK silencing. Oligonucleotides 2009 Jun;19(2):129e40.