January 2007
Volume 48, Issue 1
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Retinal Cell Biology  |   January 2007
Ornithine Transport Via Cationic Amino Acid Transporter-1 Is Involved in Ornithine Cytotoxicity in Retinal Pigment Epithelial Cells
Author Affiliations
  • Shiho Kaneko
    From the Departments of Ophthalmology and
  • Akira Ando
    From the Departments of Ophthalmology and
  • Emiko Okuda-Ashitaka
    Medical Chemistry, Kansai Medical University, Osaka, Japan; and the
  • Masahide Maeda
    Department of Regeneration and Advanced Medical Science, Graduate School of Medicine, Gifu University, Gifu, Japan.
  • Kyoji Furuta
    Department of Regeneration and Advanced Medical Science, Graduate School of Medicine, Gifu University, Gifu, Japan.
  • Masaaki Suzuki
    Department of Regeneration and Advanced Medical Science, Graduate School of Medicine, Gifu University, Gifu, Japan.
  • Miyo Matsumura
    From the Departments of Ophthalmology and
  • Seiji Ito
    Medical Chemistry, Kansai Medical University, Osaka, Japan; and the
Investigative Ophthalmology & Visual Science January 2007, Vol.48, 464-471. doi:10.1167/iovs.06-0398
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      Shiho Kaneko, Akira Ando, Emiko Okuda-Ashitaka, Masahide Maeda, Kyoji Furuta, Masaaki Suzuki, Miyo Matsumura, Seiji Ito; Ornithine Transport Via Cationic Amino Acid Transporter-1 Is Involved in Ornithine Cytotoxicity in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(1):464-471. doi: 10.1167/iovs.06-0398.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. A prior report showed ornithine cytotoxicity in ornithine-δ-aminotransferase (OAT)–deficient human retinal pigment epithelial (RPE) cells in an in vitro model of gyrate atrophy of the choroid and retina. This study was intended to clarify the mechanism of ornithine cytotoxicity and to determine the responsible amino acid transporters.

methods. The mRNA expression of amino acid transporters in human telomerase reverse transcriptase (hTERT)-RPE cells was examined by reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis. Carrier-mediated ornithine transport via the L-type amino acid transporter (LAT)1, LAT2, cationic amino acid transporter (CAT)-1, and y+LAT2 systems was evaluated by short interfering (si)RNA–mediated gene silencing. The cytoprotective effect of CAT-1-specific siRNA on ornithine cytotoxicity was measured using quantitative analysis of cellular adenosine triphosphate (ATP) at 24 hours after treatment with ornithine in OAT-deficient RPE cells.

results. LAT1, LAT2, CAT-1, and y+LAT2 mRNA expression was detected by Northern blot analysis, whereas RT-PCR revealed that LAT1, LAT2, y+LAT1, y+LAT2, CAT-1, and b0,+AT mRNAs were expressed together with the heterodimeric glycoproteins 4F2hc and rBAT in hTERT-RPE cells. l-[14C]ornithine uptake in hTERT-RPE cells was decreased by 46.6% and 22.0% by CAT-1 and y+LAT2 siRNA, respectively, whereas LAT1 and LAT2 siRNA had no significant effect. Further, CAT-1 silencing by siRNA reduced ornithine cytotoxicity in OAT-deficient RPE cells.

conclusions. The results suggest that ornithine transport via CAT-1 may play a crucial role in ornithine cytotoxicity in hTERT-RPE cells. Reduction of the ornithine transport via CAT-1 may be a new target for treatment of gyrate atrophy.

Nearly a century after the original ophthalmologic descriptions of gyrate atrophy of the choroid and retina (GA), a rare autosomal recessive chorioretinal degeneration, 1 2 Simell and Takki 3 discovered that the biochemical abnormalities of this disorder are hyperornithinemia and ornithinuria and also reported an enzyme defect, a deficiency of the mitochondrial matrix enzyme ornithine-δ-amino transferase (OAT). In patients with GA, plasma ornithine concentrations are 10- to 15-fold greater than normal, and additional abnormalities of other amino acids in the plasma such as hypolysinemia, hypoglutamic acidemia, and hypoglutaminemia are observed. Although the discovery of hyperornithinemia attracted much attention to the metabolism of ornithine in relation to the treatment of GA, there was little information on the pathogenesis of ornithine in GA. Ueda et al. 4 of our study group later reported that inactivation of OAT in human retinal pigment epithelial (RPE) cells by 5-fluoromethylornithine (5-FMO), a specific irreversible inhibitor of OAT, 5 makes them susceptible to ornithine, leading to cell death, and suggested that ornithine cytotoxicity toward OAT-deficient RPE cells by treatment with 5-FMO could be used as an in vitro model of GA. 4  
In mammalian cells, amino acids are transported through biological membranes by various transport systems, 6 7 8 with different carrier proteins that exhibit distinct transport properties participating in the amino acid transport. Cationic amino acids (CAAs), such as lysine, arginine, and histidine, are transported through the cellular membrane by four distinct transport systems: y+, y+L, b0,+, and B0,+. 9 System y+ includes CAA transporter (CAT)-1, -2A/2B, -3, and -4, which are found ubiquitously and transport CAAs specifically. System y+ is pH-independent and mediates the bidirectional transport of CAAs. 10 11 12 13 System y+L, which includes y+LAT1 and y+LAT2, is an exchangeable transporter that recognizes CAAs in the absence of sodium, though it requires the cation to interact with neutral amino acids (NAAs). 14 15 System b0,+ recognizes CAAs and NAAs with a similar affinity, and transports amino acids independent of sodium, 16 whereas system B0,+ represents an Na+/Cl-dependent transport system and transports CAAs and NAAs, with the highest affinity for hydrophobic amino acids. 9 17 Although the substrate specificity of systems b0,+ and B0,+ is similar, the latter also accepts alanine and serine. The transport of large NAAs with branched or aromatic side chains, such as leucine, isoleucine, valine, phenylalanine, tyrosine, and tryptophan, is mediated by system L, which is Na+-independent and a major route of branched or aromatic amino acids. 18 19 The amino acid transporter systems y+L, system L, cystine/glutamate transporter x c, small neutral transporter asc, and b0,+ require interaction via a disulfide bridge with the type II membrane glycoprotein members—namely, the heavy chains—for trafficking the transporters to the cell membrane. 20 System y+L, system L, x c, and asc require 4F2hc, whereas system b0,+ requires rBAT. 
Nakauchi et al. 21 have reported that both small and large NAAs and 2-amino-2-norbornane-carboxylic acid (BCH), a conventional inhibitor of system L, 22 exhibited a cytoprotective effect against ornithine cytotoxicity in OAT-deficient human telomerase reverse transcriptase (hTERT)-RPE cells. Although the mechanisms of ornithine cytotoxicity and cytoprotective effect of NAAs and BCH remain to be elucidated, these results suggest that NAAs and BCH modify ornithine transport through the cell membrane and reduce ornithine accumulation in RPE cells, resulting in a cytoprotective effect. To clarify the mechanisms of ornithine cytotoxicity and cytoprotection, we attempted to characterize the ornithine transport system in RPE cells. 
In the present study, we found that ornithine was mainly transported by CAT-1 in hTERT-RPE cells and that the suppression of CAT-1 expression by siRNA reduced ornithine cytotoxicity in OAT-deficient hTERT-RPE cells. 
Materials and Methods
Cell Culture
A human RPE cell line, hTERT-RPE, previously established by gene transfer of human telomerase reverse transcriptase cDNA, was kindly provided by Donald J. Zack (Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD). This RPE cell line is reported to have several characteristics of other normal RPE cell lines, such as expression of Rpe65 and in vitro differentiation capacity. The hTERT-RPE cells were maintained in Dulbecco’s modified Eagle’s (DME)/Ham F-12 medium (1:1; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in 5% CO2
RT-PCR and Real-Time PCR
Total RNA was isolated from an adherent monolayer of hTERT-RPE cells (TRIzol; Invitrogen-Life Technologies, Carlsbad, CA). Total RNA was reverse transcribed into cDNA using a first-strand synthesis system for RT-PCR (Invitrogen-Life Technologies). The first-strand cDNA product was amplified in a buffer containing EX-Taq DNA polymerase (Takara Bio, Ohtsu, Japan) and anti-Taq antibody (anti-Taq high; Toyobo, Osaka, Japan) with the double standard–specific fluorescent dye SYBR Green I for real-time RT-PCR, using the primer sets of LAT1, LAT2, CAT-1, CAT-2A, CAT-2B, CAT-3, CAT-4, y+LAT1, y+LAT2, b0,+AT, ATB0,+, 4F2hc, rBAT, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as shown in Table 1 . RT-PCR was performed with a PCR thermal cycler (SP; Takara Bio) with the following conditions: hold at 94°C for 30 seconds, 40 cycles of amplification (94°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute), and a final extension at 72°C for 5 minutes. Real-time quantitative RT-PCR was performed with a DNA engine real-time PCR system (Opticon; Bio-Rad Laboratories, Hercules, CA) and standard SYBR Green protocol with the following conditions: hold at 94°C for 30 seconds, 40 cycles of amplification (94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds), and a final extension at 72°C for 5 minutes. Fluorescence signals produced by binding of SYBR Green to new double-stranded amplicons were collected and analyzed after each PCR cycle with the system software (Opticon; Bio-Rad). All samples were run in triplicate. mRNA expression of each amino acid transporter was calculated based on the threshold cycle (Ct). Values were corrected by the GAPDH gene (internal control) in each experimental series. 
Aliquots (10 μL) were taken from the PCR mixtures and analyzed by electrophoresis on a 2% agarose gel in 1× Tris-acetate-EDTA buffer. The presence of the corresponding PCR products was determined by the expected sizes of the amplification products (Table 1)
Northern Blot Analysis and RNA Interference
Total RNA was isolated from hTERT-RPE cells and electrophoresed on a 1% agarose-10% formaldehyde gel, then transferred onto a nylon membrane (Schleicher & Schuell Bioscience, Keene, NH) and hybridized with 32P-labeled LAT1, LAT2, CAT-1, y+LAT1, y+LAT2, b0,+AT, and GAPDH cDNA probes. The membrane was washed twice for 10 minutes at 42°C with 2× SSPE (300 mM NaCl, 17.3 mM NaH2PO4, 2.5 mM EDTA [pH 7.4]) containing 0.1% SDS and then washed twice for 30 minutes at 42°C with 0.1× SSPE containing 0.1% SDS. After stringent washes, the membrane was exposed to x-ray film using autoradiography. For gene-silencing experiments, double-strand short interfering (si)RNAs directed against LAT1, LAT2, CAT-1, and y+LAT2 were designed and synthesized by Hokkaido System Science Co., Ltd. (Sapporo, Hokkaido, Japan). Scrambled control siRNA that had no sequence homology to any known human genes was used as the control. hTERT-RPE cells were transfected with siRNA (Lipofectamine 2000 reagent; Invitrogen-Life Technologies), according to the manufacturer’s instructions. After 5 hours of exposure, the cells were washed with DME/Ham F-12 medium supplemented with 10% FBS. After the cells were further incubated for 48 hours, total RNA was isolated, and Northern blotting was performed as just described. 
Expression Plasmids
The cDNAs of LAT1, 4F2hc, and CAT-1 were amplified by RT-PCR with cDNA synthesized from the hTERT-RPE cells. The PCR products were purified from a 1% agarose gel with a DNA extraction kit (Bio-Rad Laboratories) and subcloned into a vector (pGEM-T-easy; Promega, Madison, WI). Each sequence of the products showed 100% identity to the published sequences of LAT1, 4F2hc, and CAT1. The expression vectors were constructed by inserting coding sequences of NotI with excised LAT1, EcoRI with excised 4F2hc, and EcoRI with excised CAT-1 into pcDNA3.1. 
Measurement of [14C]ornithine and [3H]leucine Uptake
hTERT-RPE cells were seeded onto 24-well culture dishes (Falcon; Corning, Inc., Corning, NY) at a concentration of 2 × 105/well or onto permeable transwell filters (Transwell, Corning, Inc.) at a concentration of 5 × 104/well. After 24 hours, the cells were equilibrated for 30 minutes in an incubation buffer containing 125 mM NaCl, 5.6 mM d-glucose, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.3 mM CaCl2, and 25 mM HEPES (pH 7.4). For sodium dependency of ornithine uptake, NaCl was replaced by choline chloride. The uptake of l-[U-14C]ornithine (250 Ci/mol, GE Healthcare, Tokyo, Japan) and l-[4,5-3H]leucine (162 Ci/mmol, GE Healthcare) was initiated by adding 30 μL of [14C]ornithine (1 mM, 0.05 μCi) and [3H]leucine (1 mM, 5 μCi), respectively. After 2 minutes, uptake was terminated by washing the cells three times with 0.5 mL of ice-cold incubation buffer. The cells were lysed with 0.1 M NaOH containing 0.1% Triton-X, and radioactivity from the lysates was measured with a liquid scintillation counter (Tri-Carb Liquid Scintillation Analyzer 2700TR: PerkinElmer, Meriden, CT). The values are expressed as nanomoles per milligram protein per minute, and uptake linearity was retained over this period. The protein content was determined with a kit (Dc Protein Assay kit; Bio-Rad Laboratories), with bovine serum albumin as the standard. All experiments were performed at least four times and reproducible results were obtained. 
For overexpression or knockdown-expression studies, hTERT-RPE cells were seeded onto 6-cm culture dishes and transfected with each siRNA sample (Lipofectamine 2000 reagent; Invitrogen-Life Technologies) or with the expression vector and the transfection reagent, according to the manufacturer’s instructions. After 24 hours of incubation in DME/Ham F-12 medium, the cells were harvested and seeded onto 24-well culture dishes at a concentration of 2 × 105/well or seeded onto permeable transwell filters (Transwell; Corning, Inc.) at a concentration of 5 × 104/well and subjected to [14C]ornithine or [3H]leucine uptake experiments after 24-hour culture, as described earlier. 
Measurement of Ornithine Cytotoxicity
Ornithine cytotoxicity was examined as described previously. 4 Briefly, hTERT-RPE cells were transfected with control siRNA or CAT-1 siRNA at a final concentration of 20 nM. Forty-eight hours after transfection, the cells were treated with 0.5 mM of 5-FMO and 10 or 20 mM of ornithine in Ham F-12 medium for 24 hours. Ornithine cytotoxicity and the cytoprotective effect of siRNA were evaluated morphologically in micrographs taken with a digital camera (SPOT; Diagnostic Instruments, Sterling Heights, MI) through an inverted confocal microscope (IX70; Olympus, Tokyo, Japan). For quantitative examination of the prevention of ornithine cytotoxicity, we used a luminescent cell-viability assay (CellTiter-Glo; Promega), which is based on adenosine triphosphate (ATP) bioluminescence as a marker of cell viability. After 24-hour treatment with 5-FMO and/or ornithine, an equal volume of the reagent was added to each well and incubated for 10 minutes with the cells, according to the procedure recommended by the manufacturer. Luminescence produced by the luciferin+ATP reaction was measured with a spectrofluorometer (Wallac 1420 ARVOsx Multi Label Counter: PerkinElmer). Ornithine cytotoxicity was calculated as the percentage decrease of luminescence compared with the control. 
Statistics
For comparisons between multiple groups, statistical significance was determined using ANOVA with the Bonferroni correction, and for single comparisons between two groups, an unpaired Student’s t-test was used. Data are expressed as the mean ± SD of three or four separate experiments. Levels of P < 0.05 were considered statistically significant. 
Results
Effect of BCH and Leucine on Ornithine Uptake
Nakauchi et al. 21 have reported that both small and large NAAs and BCH, a conventional inhibitor of system L, 22 exhibit a cytoprotective effect against ornithine cytotoxicity in OAT-deficient hTERT-RPE cells. To clarify the mechanisms of ornithine cytotoxicity and cytoprotection, we first evaluated the effect of leucine and BCH on [14C]ornithine uptake in hTERT-RPE cells. Regardless of the presence of Na, BCH did not inhibit [14C]ornithine uptake up to 10 mM (Figs. 1A 1B) . In contrast, while [14C]ornithine uptake was reduced by 13.3% and 27.8% by leucine at 1 and 10 mM, respectively, in the Na+-free incubation buffer (Fig. 1D) , it was reduced by 28.3% by 1 mM leucine in the incubation buffer containing 125 mM NaCl (Fig. 1C) . These results suggest that [14C]ornithine uptake is mediated by amino acid transporters besides system L. 
mRNA Expression of Amino Acid Transporters in hTERT-RPE Cells
Transport of CAAs, such as arginine, lysine, and histidine, is mediated by four different transport systems in mammalian cells, but there is little information regarding CAA transport systems in RPE cells or their involvement in ornithine transport. To clarify the ornithine transport system in RPE cells, we examined the mRNA expression of CAA transporters and NAA transporter system L in hTERT-RPE cells using RT-PCR and Northern blot analysis (Fig. 2) . As shown in Figure 2A , the mRNA expression of LAT1, LAT2, y+LAT1, y+LAT2, CAT-1, b0,+AT, and their common subunits, 4F2hc and rBAT, were detected in hTERT-RPE cells as RT-PCR products with expected sizes (Table 1) . In addition, LAT1, LAT2, CAT-1, and y+LAT2 were detected by Northern blot analysis (Fig. 2B) . To clarify the discrepancy of the mRNA expression of AA transporters between RT-PCR and Northern blot analysis, we performed real-time qRT-PCR. The expression levels of AA transporters observed by Northern blot analysis were confirmed by real-time RT-PCR (Fig. 2C) . Therefore, we speculated that those four transporters detected by Northern blot analysis had a major contribution to cellular transport of amino acids and ornithine in RPE cells and chose them for examination of ornithine transport activity. 
Reduction of Ornithine Uptake by siRNA of CAT-1
To identify the amino acid transporters involved in ornithine transport, we investigated the effects of siRNA of LAT1, LAT2, CAT-1, y+LAT2, and a combination of CAT-1 plus y+LAT2 on the activity of [14C]ornithine transport in hTERT-RPE cells (Fig. 3) . Since [14C]ornithine uptake was not affected significantly by Na+ replacement with choline (data not shown), we used Na+-free buffer in the following experiments. In the hTERT-RPE cells transfected with siRNA of CAT-1 or y+LAT2 alone, mRNA expression was markedly reduced in the Northern blot analysis, whereas ornithine uptake was decreased by 46.6% and 22.0%, respectively (Figs. 3A 3B) . There was no significant difference in mRNA expression and [14C]ornithine uptake between cells transfected with and without scrambled siRNA. When the cells were transfected together with the siRNA of CAT-1 and y+LAT2, [14C]ornithine uptake was decreased by 56.5% (Fig. 3C) . Although the reduced expression of LAT1 and LAT2 mRNA was confirmed, ornithine uptake was not affected by the transfection of LAT1 or LAT2 siRNA (Figs. 3D 3E) . These results suggest that ornithine uptake may be mediated by two transporters, CAT-1 and a composite of y+LAT2 and 4F2hc, and that ornithine is mainly transported into hTERT-RPE cells by CAT-1. 
Polarity of Ornithine Uptake in CAT-1-Overexpressing RPE Cells
To clarify the transport function of LAT1 and CAT-1, we examined leucine and ornithine transport activities in hTERT-RPE cells transiently transfected with the respective expression vectors. When the cells were transfected with both pcDNA3.1-LAT1 and pcDNA3.1-4F2hc, [3H]leucine transport activity increased significantly (Fig. 4A) , though [14C]ornithine uptake did not change (Fig. 4B) . By contrast, when hTERT-RPE cells transfected with pcDNA3.1-CAT-1 were seeded onto 24-well plastic dishes, [14C]ornithine transport activity did not increase significantly (Fig. 4C) . In previous studies, CAT-1 was found to be predominantly expressed on the basolateral surface of polarized epithelial cells from the proximal tubules of the kidneys and small intestine. 24 25 26 27 To allow [14C]ornithine uptake from both apical and basolateral sides, we used permeable transwell filters (Transwell; Corning, Inc.) for the cultures. Consequently, [14C]ornithine uptake was found to be 1.8-fold higher in hTERT-RPE cells transfected with pcDNA3.1-CAT-1 (Fig. 4D) , suggesting that CAT-1 is involved in ornithine transport from the basolateral side. 
Reduction of Ornithine Cytotoxicity by siRNA of CAT-1
Ueda et al., 5 of our group, have demonstrated that inactivation of OAT in hTERT-RPE cells by 5-FMO produces ornithine cytotoxicity. To clarify whether that cytoprotective effect occurs with CAT-1 silencing, we examined the morphologic changes and cellular viability of 5-FMO-treated hTERT-RPE cells transfected with scrambled control siRNA or CAT-1 siRNA. As shown in Figure 5 , the morphologic changes in the hTERT-RPE cells at 24 hours after the addition of 10 mM ornithine were attenuated when the cells were transfected with CAT-1 siRNA. For a quantitative evaluation, we used ATP bioluminescence as a marker of cellular viability (Fig. 6) , which was calculated as the ratio of ATP bioluminescence between nontreated cells and 5-FMO- and ornithine-treated hTERT-RPE cells. After 24 hours of incubation, hTERT-RPE cells transfected with scrambled siRNA were gradually detached from dishes, and ATP bioluminescence was reduced by ornithine in a concentration-dependent manner by 19.6% and 31.1% at concentrations of 10 and 20 mM, respectively. However, ATP content was not decreased in hTERT-RPE cells transfected with CAT-1 siRNA, suggesting that OAT-deficient RPE cells were damaged by ornithine via CAT-1. 
Discussion
It has been reported that an arginine-restricted diet is effective for treatment of patients with GA and that RPE degeneration is slowed by a low-arginine diet in OAT knockout mice. 2 Arginine restriction may correct a high blood ornithine level by inhibition of the production of ornithine and inhibit progression of the disease process. We speculated that chorioretinal degeneration in patients with GA would not develop if ornithine was not transported into RPE cells. Further, we thought that the hTERT-RPE cells in the GA model previously established by our group might provide an OAT-deficient GA model of RPE cells similar to the cells found in patients with GA and OAT knockout mice. 4  
RPE cells did not exhibit ornithine susceptibility until OAT was inactivated in the present model, which suggests that sensitivity for ornithine was increased by that inactivation. A previous study conducted by our group showed that NAAs, including leucine and BCH, an inhibitor of NAA transporter system L, relieved ornithine cytotoxicity in ornithine-susceptible RPE cells by inactivation of OAT. 22 Further, the transport of ornithine into RPE cells was shown to be decreased by the addition of leucine. These results imply that even if ornithine-susceptible RPE cells are exposed to ornithine at a high concentration, the cells are not damaged if ornithine is not transported and/or accumulated intracellularly. 
It has been reported that CAT-1, y+LAT1, and b0,+AT recognize ornithine as their substrate and transport it when they are expressed in Xenopus oocytes, though the contribution of their transport systems to ornithine transport in RPE cells remains largely unknown. In the present study, we investigated the expression patterns of amino acid transporters in hTERT-RPE cells and attempted to identify which transporter is the main contributor to ornithine transport. LAT1, LAT2, y+LAT1, y+LAT2, CAT-1, and b0,+AT mRNA expression in hTERT-RPE cells was observed by RT-PCR (Fig. 2A) . However, because the expression level of y+LAT1 and b0,+AT was faint or undetectable in our Northern blot analysis (Fig. 2B) , we concluded that y+LAT1 and b0,+AT made little contribution to the cellular transport of ornithine. In addition, there was only a slight sodium ion dependency in ornithine transport in hTERT-RPE cells (<8%, data not shown), suggesting that there was little contribution by Na+-dependent amino acid transporters such as system B0,+. We also found that the uptake of ornithine was decreased by 46.6% and 22.0% by siRNA for CAT-1 and y+LAT2, respectively (Fig. 3)
In addition, we measured ornithine transport activity in hTERT-RPE cells overexpressing LAT1 or CAT-1. In LAT1/4F2hc–overexpressing cells, leucine uptake was significantly increased (Fig. 4A) , whereas ornithine transport was not affected (Fig. 4B) . In hTERT-RPE cells transfected with CAT-1 for transport study, ornithine transport activity was not changed when the cells were seeded onto plastic dishes (Fig. 4C) ; however, when grown on the microporous filters (Transwell; Corning, Inc.), in which the cells took up ornithine from both the apical and basolateral sides, ornithine transport activity was significantly increased (Fig. 4D) . These results are consistent with the previous observation that CAT-1 amino acid transporters are found more frequently on the basolateral sides of cellular membranes with polarized epithelial cells. 24 25 26 27 According to another report, system y+ has a lower affinity to CAAs such as arginine and lysine (K m, 70–250 μM), and its specificity is restricted to CAAs, whereas system y+L has a higher affinity to CAAs (K m, 6–10 μM) in the absence of Na+ and can also transport NAAs in the presence of Na+. 8 At low substrate concentrations, both systems may contribute to the influx of ornithine similarly; however, at 1 mM, the activity of system y+, which has a 10-fold higher V max than does system y+L, exceeds that of system y+L. 28 29 Together with the present observation that ornithine transport activity did not increase significantly in y+LAT2/4F2hc overexpressing cells (data not shown), these results suggest that CAT-1 is the main ornithine transporter in hTERT-RPE cells. 
Nakauchi et al. 21 have reported that NAAs such as leucine and BCH, a conventional inhibitor of system L, 22 exhibit a cytoprotective effect against ornithine cytotoxicity in OAT-deficient hTERT-RPE cells. Because substrates of LAT including leucine reduced ornithine accumulation, we speculated that LAT1 and/or LAT2 may contribute to ornithine transport. As shown in Figures 1A and 1B , however, BCH did not inhibit ornithine uptake, demonstrating that ornithine was not transported by system L. Consistent with the results obtained by Nakauchi et al., 21 leucine inhibited [14C]ornithine uptake by 28.3% at 1 mM in the incubation buffer containing NaCl (Fig. 1C) , whereas the inhibitory effect of leucine was 13.3% and 27.8% at 1 and 10 mM, respectively, in an Na+-free buffer (Fig. 1D) . Furthermore, we could not detect ornithine transport activity in either LAT1 or LAT2 (Figs. 3D 3E) , which suggested that NAAs and BCH indirectly attenuate the intracellular accumulation of ornithine. The NAA and CAA transporter y+LAT exchanges amino acids inside and outside of cells at a ratio of 1:1. Under physiological conditions, the inwardly directed sodium gradient favors efflux of intracellular CAAs in exchange for the entry of NAAs and sodium. Therefore, it seems that system y+L serves essentially as an efflux pathway for ornithine. The cooperation of plural amino acid transporters has been reported: b0,+AT cooperates with y+LAT1 or LAT2 in MDCK cells. 30 Therefore, it is possible that CAA efflux and NAA influx through y+LAT is increased, together with the inhibition of neutral amino acid transport via LAT by the addition of a neutral amino acid or BCH, resulting in a decrease in intracellular ornithine level and relief of cell damage. 
In the present study, ornithine cytotoxicity was decreased and cellular viability, examined with an ATP bioluminescence assay, was improved by the suppression of CAT-1 expression. Although additional investigations are needed to clarify the mechanisms of ornithine cytotoxicity and the cytoprotective effect of leucine in RPE cells, the present results clearly demonstrated that ornithine is mainly transported into hTERT-RPE cells via CAT-1 and that suppression of CAT-1 expression decreases the intracellular level of ornithine and protects against ornithine cytotoxicity. It has been reported that the transport of CAAs into most mammalian cells is mediated mainly by system y+ 31 and that CAT-1 interacts with endothelial nitric oxide synthase. 32 33 Thus, CAT-1 may be crucial for the maintenance of normal cellular function. In addition, CAT-1 was first described as a cell-surface receptor for ecotropic murine retroviruses. 10 11 12 Therefore, there may be problems with clinical attempts to suppress CAT-1 expression, due to the inhibition of normal cellular function. If the expression level of CAT-1 or ornithine uptake via CAT-1 in only RPE cells could be controlled by topical application, a decrease in the uptake of ornithine into RPE cells may be a good strategy for the treatment of GA, in addition to correction of high blood ornithine levels by dietary restriction. 
 
Table 1.
 
Primer Sequences for RT-PCR Analysis
Table 1.
 
Primer Sequences for RT-PCR Analysis
Gene Primer Product Size (bp)
CAT-1 Forward 5′-TGCGCTCTTTCCGCCAGTCT-3′ 351
Reverse 5′-GGTGCTTGCCAATTCATTTT-3′
CAT-2A Forward 5′-GTTGACTGCAGGGGTCATTT-3′ 163
Reverse 5′-ACATTTGGGCTGGTCGTAAG-3′
CAT-2B Forward 5′-GAGGATGGGTTGCTTTTCAA-3′ 232
Reverse 5′-ACATTTGGGCTGGTCGTAAG-3′
CAT-3 Forward 5′-CATTGGTACAGCCAGTGTGGC-3′ 325
Reverse 5′-GTTCAGCCATGGCCAATTCGTAG-3′
CAT-4 Forward 5′-ATGGGTACAGCCCTGGAGCA-3′ 338
Reverse 5′-TACACTGCAAGTCCCATCAG-3′
y+LAT1 Forward 5′-ACTGTGCCTATGTCAAATGGGGAAC-3′ 305
Reverse 5′-ATAGATGATGGTGACAATGGGC-3′
y+LAT2 Forward 5′-TAAACTGTGCCAGGGACACT-3′ 299
Reverse 5′-TCTGGTCAGCAAATGTCACA-3′
b0,+AT Forward 5′-AGCTTGGCACAATGATCACCAAGTC-3′ 327
Reverse 5′-TGATGATGATGATGGCCACGATCAC-3′
ATB0,+ Forward 5′-GCAATATTTATCTGGTCATTGGTGC-3′ 302
Reverse 5′-CTGCTGCCACTAACAGTAGGTATTT-3′
LAT1 Forward 5′-ATTATACAGCGGCCTCTTTGCCTATG-3′ 306
Reverse 5′-TGGAGGATGTGAACAGGGACCCATT-3′
LAT2 Forward 5′-ACCGAAACAACACCGAAAAG-3′ 203
Reverse 5′-CAATCCAGACGATGAGAGCA-3′
4F2hc Forward 5′-TGAATGAGTTAGAGCCCGAGAAGCA-3′ 308
Reverse 5′-CTTCTGCGCCGGTAGCTCGCGACAA-3′
rBAT Forward 5′-GGGAACAGCGTGTATGAGGT-3′ 166
Reverse 5′-GGAGTTCCAGGGAGTGTGAA-3′
GAPDH Forward 5′-CGACCACTTTGTCAAGCTCA-3′ 228
Reverse 5′-AGGGGTCTACATGGCAACTG-3′
Figure 1.
 
Effects of BCH and leucine on [14C]ornithine uptake in hTERT-RPE cells. hTERT-RPE cells (2 × 105/well) seeded on 24-well plastic dishes were cultured in DME/Ham F-12 medium (1:1) supplemented with 10% FBS 24 hours before the experiments. The uptake of [14C]ornithine (1 mM, 0.05 μCi/well) in hTERT-RPE cells was measured at 37°C for 2 minutes in the presence or absence of BCH (A, B) or leucine (C, D). For assessment of Na+-dependency, the uptake was determined in the incubation buffer containing 125 mM NaCl (A, C) or choline chloride instead of NaCl (B, D). The data shown represent the mean ± SD of results in four experiments. *P < 0.05, **P < 0.01 compared with the control.
Figure 1.
 
Effects of BCH and leucine on [14C]ornithine uptake in hTERT-RPE cells. hTERT-RPE cells (2 × 105/well) seeded on 24-well plastic dishes were cultured in DME/Ham F-12 medium (1:1) supplemented with 10% FBS 24 hours before the experiments. The uptake of [14C]ornithine (1 mM, 0.05 μCi/well) in hTERT-RPE cells was measured at 37°C for 2 minutes in the presence or absence of BCH (A, B) or leucine (C, D). For assessment of Na+-dependency, the uptake was determined in the incubation buffer containing 125 mM NaCl (A, C) or choline chloride instead of NaCl (B, D). The data shown represent the mean ± SD of results in four experiments. *P < 0.05, **P < 0.01 compared with the control.
Figure 2.
 
mRNA expression of amino acid transporters in hTERT-RPE cells. Total RNA was extracted from hTERT-RPE cells and analyzed by RT-PCR (A), Northern blot analysis (B), and real-time quantitative PCR (C). (A) Total RNA (1 μg) was reverse transcribed and amplified with primer sets of NAA and CAA transporters (Table 1) , with GAPDH used as the control. PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. (B) Total RNA (10 μg/lane) was separated on 1% agarose-10% formaldehyde gels and transferred to a nylon membrane. The membrane was hybridized with 32P-labeled probes, with GAPDH used as the control. (C) A comparison of threshold cycles using a real-time PCR method. Values were corrected by the GAPDH gene (internal control) in each experimental series.
Figure 2.
 
mRNA expression of amino acid transporters in hTERT-RPE cells. Total RNA was extracted from hTERT-RPE cells and analyzed by RT-PCR (A), Northern blot analysis (B), and real-time quantitative PCR (C). (A) Total RNA (1 μg) was reverse transcribed and amplified with primer sets of NAA and CAA transporters (Table 1) , with GAPDH used as the control. PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. (B) Total RNA (10 μg/lane) was separated on 1% agarose-10% formaldehyde gels and transferred to a nylon membrane. The membrane was hybridized with 32P-labeled probes, with GAPDH used as the control. (C) A comparison of threshold cycles using a real-time PCR method. Values were corrected by the GAPDH gene (internal control) in each experimental series.
Figure 3.
 
RNA interference of CAT-1, y+LAT2, LAT1, and LAT2 mRNA expression and ornithine transport activity in siRNA-transfected hTERT-RPE cells. Cells (7.5 × 105 /well) were transfected without siRNA, with 20 nM of scrambled siRNA, or 20 nM of siRNA of CAT-1 (A), y+LAT2 (B), CAT-1+ y+LAT2 (C), LAT1 (D), and LAT2 (E). Total RNA was isolated at 48 hours after transfection and Northern blot analysis was performed. The uptake of [14C]ornithine (1 mM, 0.05 μCi) was measured at 37°C for 2 minutes at 48 hours after siRNA transfection. The data shown represent the mean ± SD of results in four independent experiments. **P < 0.01 compared with control siRNA.
Figure 3.
 
RNA interference of CAT-1, y+LAT2, LAT1, and LAT2 mRNA expression and ornithine transport activity in siRNA-transfected hTERT-RPE cells. Cells (7.5 × 105 /well) were transfected without siRNA, with 20 nM of scrambled siRNA, or 20 nM of siRNA of CAT-1 (A), y+LAT2 (B), CAT-1+ y+LAT2 (C), LAT1 (D), and LAT2 (E). Total RNA was isolated at 48 hours after transfection and Northern blot analysis was performed. The uptake of [14C]ornithine (1 mM, 0.05 μCi) was measured at 37°C for 2 minutes at 48 hours after siRNA transfection. The data shown represent the mean ± SD of results in four independent experiments. **P < 0.01 compared with control siRNA.
Figure 4.
 
l-[3H]leucine and [14C]ornithine uptake in LAT1 and CAT-1-overexpressing hTERT-RPE cells. The uptake of (A) [3H]leucine (1 mM, 5 μCi) and (B) [14C]ornithine (1 mM, 0.05 μCi) was measured in hTERT-RPE cells transfected with pCMV-LAT1 and pCMV-4F2hc. [14C]ornithine uptake was measured in CAT-1–transfected hTERT-RPE cells (2 × 105/well) grown in 24-well plastic dishes (C) and permeable transwell filters (D). All experiments were performed at 37°C for 2 minutes at 48 hours after transfection. The data shown represent the mean ± SD of results in four experiments. **P < 0.01 compared with empty vector.
Figure 4.
 
l-[3H]leucine and [14C]ornithine uptake in LAT1 and CAT-1-overexpressing hTERT-RPE cells. The uptake of (A) [3H]leucine (1 mM, 5 μCi) and (B) [14C]ornithine (1 mM, 0.05 μCi) was measured in hTERT-RPE cells transfected with pCMV-LAT1 and pCMV-4F2hc. [14C]ornithine uptake was measured in CAT-1–transfected hTERT-RPE cells (2 × 105/well) grown in 24-well plastic dishes (C) and permeable transwell filters (D). All experiments were performed at 37°C for 2 minutes at 48 hours after transfection. The data shown represent the mean ± SD of results in four experiments. **P < 0.01 compared with empty vector.
Figure 5.
 
Inhibition of ornithine cytotoxicity by CAT-1 silencing in 5-FMO-treated hTERT-RPE cells. hTERT-RPE cells (7.5 × 105 /well) were transfected with 20 nM of scrambled siRNA or 20 nM of CAT-1 siRNA, then incubated with or without 0.5 mM 5-FMO and 10 mM ornithine. Morphologic change was assessed with a confocal microscope. Original magnification, ×100.
Figure 5.
 
Inhibition of ornithine cytotoxicity by CAT-1 silencing in 5-FMO-treated hTERT-RPE cells. hTERT-RPE cells (7.5 × 105 /well) were transfected with 20 nM of scrambled siRNA or 20 nM of CAT-1 siRNA, then incubated with or without 0.5 mM 5-FMO and 10 mM ornithine. Morphologic change was assessed with a confocal microscope. Original magnification, ×100.
Figure 6.
 
Cellular viability of CAT-1 siRNA-transfected hTERT-RPE cells. Forty-eight hours after transfection with 20 nM of scrambled control siRNA or 20 nM of CAT-1 siRNA, the cells were incubated for 24 hours with 0.5 mM 5-FMO, 10 mM ornithine, 0.5 mM 5-FMO, and 10 mM ornithine or 0.5 mM 5-FMO and 20 mM ornithine in Ham F-12 medium. The ATP content in the cells was measured, to determine the number of metabolically active cells. The data represent the mean ± SD of results in three experiments. **P < 0.01 compared with the control.
Figure 6.
 
Cellular viability of CAT-1 siRNA-transfected hTERT-RPE cells. Forty-eight hours after transfection with 20 nM of scrambled control siRNA or 20 nM of CAT-1 siRNA, the cells were incubated for 24 hours with 0.5 mM 5-FMO, 10 mM ornithine, 0.5 mM 5-FMO, and 10 mM ornithine or 0.5 mM 5-FMO and 20 mM ornithine in Ham F-12 medium. The ATP content in the cells was measured, to determine the number of metabolically active cells. The data represent the mean ± SD of results in three experiments. **P < 0.01 compared with the control.
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Figure 1.
 
Effects of BCH and leucine on [14C]ornithine uptake in hTERT-RPE cells. hTERT-RPE cells (2 × 105/well) seeded on 24-well plastic dishes were cultured in DME/Ham F-12 medium (1:1) supplemented with 10% FBS 24 hours before the experiments. The uptake of [14C]ornithine (1 mM, 0.05 μCi/well) in hTERT-RPE cells was measured at 37°C for 2 minutes in the presence or absence of BCH (A, B) or leucine (C, D). For assessment of Na+-dependency, the uptake was determined in the incubation buffer containing 125 mM NaCl (A, C) or choline chloride instead of NaCl (B, D). The data shown represent the mean ± SD of results in four experiments. *P < 0.05, **P < 0.01 compared with the control.
Figure 1.
 
Effects of BCH and leucine on [14C]ornithine uptake in hTERT-RPE cells. hTERT-RPE cells (2 × 105/well) seeded on 24-well plastic dishes were cultured in DME/Ham F-12 medium (1:1) supplemented with 10% FBS 24 hours before the experiments. The uptake of [14C]ornithine (1 mM, 0.05 μCi/well) in hTERT-RPE cells was measured at 37°C for 2 minutes in the presence or absence of BCH (A, B) or leucine (C, D). For assessment of Na+-dependency, the uptake was determined in the incubation buffer containing 125 mM NaCl (A, C) or choline chloride instead of NaCl (B, D). The data shown represent the mean ± SD of results in four experiments. *P < 0.05, **P < 0.01 compared with the control.
Figure 2.
 
mRNA expression of amino acid transporters in hTERT-RPE cells. Total RNA was extracted from hTERT-RPE cells and analyzed by RT-PCR (A), Northern blot analysis (B), and real-time quantitative PCR (C). (A) Total RNA (1 μg) was reverse transcribed and amplified with primer sets of NAA and CAA transporters (Table 1) , with GAPDH used as the control. PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. (B) Total RNA (10 μg/lane) was separated on 1% agarose-10% formaldehyde gels and transferred to a nylon membrane. The membrane was hybridized with 32P-labeled probes, with GAPDH used as the control. (C) A comparison of threshold cycles using a real-time PCR method. Values were corrected by the GAPDH gene (internal control) in each experimental series.
Figure 2.
 
mRNA expression of amino acid transporters in hTERT-RPE cells. Total RNA was extracted from hTERT-RPE cells and analyzed by RT-PCR (A), Northern blot analysis (B), and real-time quantitative PCR (C). (A) Total RNA (1 μg) was reverse transcribed and amplified with primer sets of NAA and CAA transporters (Table 1) , with GAPDH used as the control. PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. (B) Total RNA (10 μg/lane) was separated on 1% agarose-10% formaldehyde gels and transferred to a nylon membrane. The membrane was hybridized with 32P-labeled probes, with GAPDH used as the control. (C) A comparison of threshold cycles using a real-time PCR method. Values were corrected by the GAPDH gene (internal control) in each experimental series.
Figure 3.
 
RNA interference of CAT-1, y+LAT2, LAT1, and LAT2 mRNA expression and ornithine transport activity in siRNA-transfected hTERT-RPE cells. Cells (7.5 × 105 /well) were transfected without siRNA, with 20 nM of scrambled siRNA, or 20 nM of siRNA of CAT-1 (A), y+LAT2 (B), CAT-1+ y+LAT2 (C), LAT1 (D), and LAT2 (E). Total RNA was isolated at 48 hours after transfection and Northern blot analysis was performed. The uptake of [14C]ornithine (1 mM, 0.05 μCi) was measured at 37°C for 2 minutes at 48 hours after siRNA transfection. The data shown represent the mean ± SD of results in four independent experiments. **P < 0.01 compared with control siRNA.
Figure 3.
 
RNA interference of CAT-1, y+LAT2, LAT1, and LAT2 mRNA expression and ornithine transport activity in siRNA-transfected hTERT-RPE cells. Cells (7.5 × 105 /well) were transfected without siRNA, with 20 nM of scrambled siRNA, or 20 nM of siRNA of CAT-1 (A), y+LAT2 (B), CAT-1+ y+LAT2 (C), LAT1 (D), and LAT2 (E). Total RNA was isolated at 48 hours after transfection and Northern blot analysis was performed. The uptake of [14C]ornithine (1 mM, 0.05 μCi) was measured at 37°C for 2 minutes at 48 hours after siRNA transfection. The data shown represent the mean ± SD of results in four independent experiments. **P < 0.01 compared with control siRNA.
Figure 4.
 
l-[3H]leucine and [14C]ornithine uptake in LAT1 and CAT-1-overexpressing hTERT-RPE cells. The uptake of (A) [3H]leucine (1 mM, 5 μCi) and (B) [14C]ornithine (1 mM, 0.05 μCi) was measured in hTERT-RPE cells transfected with pCMV-LAT1 and pCMV-4F2hc. [14C]ornithine uptake was measured in CAT-1–transfected hTERT-RPE cells (2 × 105/well) grown in 24-well plastic dishes (C) and permeable transwell filters (D). All experiments were performed at 37°C for 2 minutes at 48 hours after transfection. The data shown represent the mean ± SD of results in four experiments. **P < 0.01 compared with empty vector.
Figure 4.
 
l-[3H]leucine and [14C]ornithine uptake in LAT1 and CAT-1-overexpressing hTERT-RPE cells. The uptake of (A) [3H]leucine (1 mM, 5 μCi) and (B) [14C]ornithine (1 mM, 0.05 μCi) was measured in hTERT-RPE cells transfected with pCMV-LAT1 and pCMV-4F2hc. [14C]ornithine uptake was measured in CAT-1–transfected hTERT-RPE cells (2 × 105/well) grown in 24-well plastic dishes (C) and permeable transwell filters (D). All experiments were performed at 37°C for 2 minutes at 48 hours after transfection. The data shown represent the mean ± SD of results in four experiments. **P < 0.01 compared with empty vector.
Figure 5.
 
Inhibition of ornithine cytotoxicity by CAT-1 silencing in 5-FMO-treated hTERT-RPE cells. hTERT-RPE cells (7.5 × 105 /well) were transfected with 20 nM of scrambled siRNA or 20 nM of CAT-1 siRNA, then incubated with or without 0.5 mM 5-FMO and 10 mM ornithine. Morphologic change was assessed with a confocal microscope. Original magnification, ×100.
Figure 5.
 
Inhibition of ornithine cytotoxicity by CAT-1 silencing in 5-FMO-treated hTERT-RPE cells. hTERT-RPE cells (7.5 × 105 /well) were transfected with 20 nM of scrambled siRNA or 20 nM of CAT-1 siRNA, then incubated with or without 0.5 mM 5-FMO and 10 mM ornithine. Morphologic change was assessed with a confocal microscope. Original magnification, ×100.
Figure 6.
 
Cellular viability of CAT-1 siRNA-transfected hTERT-RPE cells. Forty-eight hours after transfection with 20 nM of scrambled control siRNA or 20 nM of CAT-1 siRNA, the cells were incubated for 24 hours with 0.5 mM 5-FMO, 10 mM ornithine, 0.5 mM 5-FMO, and 10 mM ornithine or 0.5 mM 5-FMO and 20 mM ornithine in Ham F-12 medium. The ATP content in the cells was measured, to determine the number of metabolically active cells. The data represent the mean ± SD of results in three experiments. **P < 0.01 compared with the control.
Figure 6.
 
Cellular viability of CAT-1 siRNA-transfected hTERT-RPE cells. Forty-eight hours after transfection with 20 nM of scrambled control siRNA or 20 nM of CAT-1 siRNA, the cells were incubated for 24 hours with 0.5 mM 5-FMO, 10 mM ornithine, 0.5 mM 5-FMO, and 10 mM ornithine or 0.5 mM 5-FMO and 20 mM ornithine in Ham F-12 medium. The ATP content in the cells was measured, to determine the number of metabolically active cells. The data represent the mean ± SD of results in three experiments. **P < 0.01 compared with the control.
Table 1.
 
Primer Sequences for RT-PCR Analysis
Table 1.
 
Primer Sequences for RT-PCR Analysis
Gene Primer Product Size (bp)
CAT-1 Forward 5′-TGCGCTCTTTCCGCCAGTCT-3′ 351
Reverse 5′-GGTGCTTGCCAATTCATTTT-3′
CAT-2A Forward 5′-GTTGACTGCAGGGGTCATTT-3′ 163
Reverse 5′-ACATTTGGGCTGGTCGTAAG-3′
CAT-2B Forward 5′-GAGGATGGGTTGCTTTTCAA-3′ 232
Reverse 5′-ACATTTGGGCTGGTCGTAAG-3′
CAT-3 Forward 5′-CATTGGTACAGCCAGTGTGGC-3′ 325
Reverse 5′-GTTCAGCCATGGCCAATTCGTAG-3′
CAT-4 Forward 5′-ATGGGTACAGCCCTGGAGCA-3′ 338
Reverse 5′-TACACTGCAAGTCCCATCAG-3′
y+LAT1 Forward 5′-ACTGTGCCTATGTCAAATGGGGAAC-3′ 305
Reverse 5′-ATAGATGATGGTGACAATGGGC-3′
y+LAT2 Forward 5′-TAAACTGTGCCAGGGACACT-3′ 299
Reverse 5′-TCTGGTCAGCAAATGTCACA-3′
b0,+AT Forward 5′-AGCTTGGCACAATGATCACCAAGTC-3′ 327
Reverse 5′-TGATGATGATGATGGCCACGATCAC-3′
ATB0,+ Forward 5′-GCAATATTTATCTGGTCATTGGTGC-3′ 302
Reverse 5′-CTGCTGCCACTAACAGTAGGTATTT-3′
LAT1 Forward 5′-ATTATACAGCGGCCTCTTTGCCTATG-3′ 306
Reverse 5′-TGGAGGATGTGAACAGGGACCCATT-3′
LAT2 Forward 5′-ACCGAAACAACACCGAAAAG-3′ 203
Reverse 5′-CAATCCAGACGATGAGAGCA-3′
4F2hc Forward 5′-TGAATGAGTTAGAGCCCGAGAAGCA-3′ 308
Reverse 5′-CTTCTGCGCCGGTAGCTCGCGACAA-3′
rBAT Forward 5′-GGGAACAGCGTGTATGAGGT-3′ 166
Reverse 5′-GGAGTTCCAGGGAGTGTGAA-3′
GAPDH Forward 5′-CGACCACTTTGTCAAGCTCA-3′ 228
Reverse 5′-AGGGGTCTACATGGCAACTG-3′
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