UbiA prenyltransferase domain–containing protein-1 modulates HMG-CoA reductase degradation to coordinate synthesis of sterol and nonsterol isoprenoids

UBIAD1 (UbiA prenyltransferase domain–containing protein-1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize vitamin K2. We previously reported that sterols stimulate binding of UBIAD1 to endoplasmic reticulum (ER)–localized 3-hydroxy-3-methylglutaryl (HMG) CoA reductase. UBIAD1 binding inhibits sterol-accelerated, ER-associated degradation (ERAD) of reductase, one of several mechanisms for feedback control of this rate-limiting enzyme in the branched pathway that produces cholesterol and nonsterol isoprenoids such as GGpp. Accumulation of GGpp in ER membranes triggers release of UBIAD1 from reductase, permitting its maximal ERAD and ER-to-Golgi transport of UBIAD1. Mutant UBIAD1 variants associated with Schnyder corneal dystrophy (SCD), a human disorder characterized by corneal accumulation of cholesterol, resist GGpp-induced release from reductase and remain sequestered in the ER to block reductase ERAD. Using lines of genetically manipulated cells, we now examine consequences of UBIAD1 deficiency and SCD-associated UBIAD1 on reductase ERAD and cholesterol synthesis. Our results indicated that reductase becomes destabilized in the absence of UBIAD1, resulting in reduced cholesterol synthesis and intracellular accumulation. In contrast, an SCD-associated UBIAD1 variant inhibited reductase ERAD, thereby stabilizing the enzyme and contributing to enhanced synthesis and intracellular accumulation of cholesterol. Finally, we present evidence that GGpp-regulated, ER-to-Golgi transport enables UBIAD1 to modulate reductase ERAD such that synthesis of nonsterol isoprenoids is maintained in sterol-replete cells. These findings further establish UBIAD1 as a central player in the reductase ERAD pathway and regulation of isoprenoid synthesis. They also indicate that UBIAD1-mediated inhibition of reductase ERAD underlies cholesterol accumulation associated with SCD.

ferase domain-containing protein-1) (12), an integral membrane prenyltransferase that utilizes GGpp to synthesize the vitamin K 2 subtype menaquinone-4 (MK-4) (13). GGpp triggers release of UBIAD1 from reductase, allowing for the maximal ERAD of reductase and ER-to-Golgi transport of UBIAD1. Eliminating expression of UBIAD1 relieves the GGpp requirement for reductase ERAD, indicating that the reaction is inhibited by the prenyltransferase. Missense mutations in the UBIAD1 gene cause Schnyder corneal dystrophy (SCD), an autosomal eye disease in humans caused by abnormal accumulation of cholesterol in the cornea (14,15). SCD-associated mutants of UBIAD1 resist GGpp-induced release from reductase and remain sequestered in the ER where they inhibit reductase ERAD in a dominant-negative fashion (12,16). Utilizing cells genetically manipulated using CRISPR/Cas9 techniques, we determine in the current studies the consequences of UBIAD1 deficiency and SCD-associated UBIAD1 on regulation of the mevalonate pathway. The results of these studies not only further establish a key role for UBIAD1 in regulation of reductase ERAD and mevalonate metabolism, but they also indicate that inhibition of reductase ERAD significantly contributes to accumulation of cholesterol associated with SCD.
We next sought to determine the consequence of UBIAD1 deficiency and reduced expression of reductase on synthesis of cholesterol from the two-carbon precursor acetate. As shown in the experiment of Fig. 2A, SV-589 (⌬UBIAD1) cells incorporated significantly less [ 14 C]acetate into cholesterol than parental SV-589 cells when cultured in FCS-containing medium. To determine whether exogenous cholesterol from FCS-derived lipoproteins contributed to reduced synthesis of cholesterol in SV-589 (⌬UBIAD1) cells, we measured incorporation of [ 3 H]mevalonate into cholesterol in wild-type and UBIAD1-deficient cells. The results show that incorporation of [ 3 H]mevalonate into cholesterol was reduced 3-4-fold in SV-589 (⌬UBIAD1) cells cultured in FCS compared with that in SV-589 cells (Fig. 2B). However, this difference was ablated when the cells were cultured in LPDS. This result indicates that reduced expression of reductase renders SV-589 (⌬UBIAD1) cells reliant on exogenous lipoproteins to satisfy cellular demands for cholesterol. The uptake of exogenous lipoproteins likely reduced incorporation of [ 3 H]mevalonate into cholesterol because of modulation of the activation of sterol regulatory element-binding protein (SREBP)-2, a membrane-bound transcription factor that controls expression of genes encoding reductase and other enzymes of the mevalonate pathway (17).
The synthesis of cholesteryl esters, the major storage form of cellular cholesterol, was next measured in wild-type and UBIAD1-deficient SV-589 cells (Fig. 4A). Similar to results obtained for cholesterol synthesis (Fig. 3A)  cells/60-mm dish in medium A containing 10% FCS and 1 mM mevalonate. On day 1, the cells were refed medium A supplemented with either 10% FCS or LPDS as indicated. After 16 h at 37°C, the cells were harvested for subcellular fractionation (A) or preparation of total RNA (B) as described under "Experimental procedures." A, aliquots of membrane fractions (20 g protein/lane) were subjected to SDS-PAGE, and immunoblot analysis was carried with IgG-A9 (against HMG CoA reductase), IgG-H8 (against endogenous UBIAD1), IgG-D1 (against ACAT-1), and anti-calnexin IgG. B, total RNA was subjected to quantitative RT-PCR using primers against human reductase; the human 36B4 mRNA was used as an invariant control. Each value represents the amount of reductase mRNA relative to that in SV-589 cells, which is arbitrarily defined as 1. C, SV-589(⌬UBIAD1)/pMyc-UBIAD1 (WT) and SV-589(⌬UBIAD1)/pMyc-UBIAD1 (N102S) cells were set up on day 0 at 3 ϫ 10 5 cells/60-mm dish in medium A containing 10% FCS. On day 1, the cells were refed identical medium and incubated for 16 h at 37°C. The cells were then fixed and analyzed by immunofluorescence microscopy using IgG-9E10 (against Myc-UBIAD1) using a Zeiss Axio Observer Epifluorescence microscope as described under "Experimental procedures. Considering our previous results that implicated UBIAD1 as a novel sensor of GGpp embedded in membranes (16) and the reciprocal synthesis of sterol and nonsterol isoprenoids in sterol-replete cells (3,18), we next compared the ability of mevalonate to restore Golgi localization of endogenous UBIAD1 in statin-treated cells cultured in LPDS and FCS. When SV-589 cells were depleted of exogenous sterols through incubation in LPDS-containing medium, UBIAD1 localized to the Golgi as expected (Fig. 5A, panel 1). Treatment of the cells with com-

UBIAD1 coordinates sterol and nonsterol isoprenoid synthesis
pactin to deplete nonsterol isoprenoids disrupted Golgi localization of UBIAD1, causing it to accumulate in the ER (panel 2). A high concentration of mevalonate (10 mM) was required to stimulate transport of UBIAD1 from the ER to Golgi in the sterol and nonsterol isoprenoid-depleted cells (panels 3-6). The Golgi localization of UBIAD1 was also disrupted by compactin when SV-589 cells were grown in lipoprotein-rich FCS-containing medium (Fig. 5A, compare panels 7 and 8). However, Golgi transport of UBIAD1 was triggered by lower concentrations of mevalonate (1-3 mM) when SV-589 cells were cultured under sterol-replete conditions (panels [10][11][12]. Similar results were obtained when isoprenoid-depleted SV-589 cells were treated with 25-HC rather than FCS for repletion of sterols. As shown in Fig. 5B, Golgi localization of UBIAD1 was restored by lower concentrations of mevalonate when cells were incubated with 25-HC (panels 10-12) compared with those required in the absence of the oxysterol (panels 4-6).
We next measured the synthesis of MK-4 in cells using a recently established assay in our group. SV-589 cells were deprived of isoprenoids through incubation in medium containing LPDS and compactin. In addition, the cells received In sterol-deprived cells, transcriptionally active fragments of SREBP-2 become proteolytically released from Golgi membranes (17,19). Once released, these fragments migrate to the nucleus, where they modulate transcription of genes required for the synthesis of cholesterol (17). Sterol accumulation inhibits proteolytic release of SREBP-2 from membranes, which results in the decline in transcription of SREBP-2 target genes and the rate of cholesterol synthesis. Quantitative RT-PCR reveals that expression of mRNAs encoding reductase and other cholesterol biosynthetic enzymes including HMG CoA synthase (HMGCS), Fpp synthase, and squalene synthase (SQS) were markedly reduced by 25-HC (Fig. 6B). In contrast, levels of mRNAs for geranylgeranyl pyrophosphate synthase (GGPPS) and UBIAD1 remained constant regardless of the absence or presence of compactin, mevalonate, or 25-HC. Immunoblot analysis revealed a reduction in the protein levels for HMGCS, transcriptionally active nuclear SREBP-2, reductase, and SQS in 25-HC treated cells, whereas GGPPS and UBIAD1 protein levels were unchanged (Fig. 6C).

Discussion
The current results provide further evidence for a pivotal role of UBIAD1 in the regulation of reductase ERAD and metabo-lism of mevalonate. This evidence includes results showing that in the absence of UBIAD1, ERAD of reductase was accelerated (Fig. 1A), causing a fall in the synthesis of cholesterol ( Fig. 2A) and cholesteryl esters (Fig. 4A). As a result, UBIAD1-deficient cells exhibited reduced storage of cholesteryl esters and other neutral lipids compared with wild-type cells as indicated by oil red O staining (Fig. 4, C and D). Nickerson et al. (20) reported an association of UBIAD1 with the ER enzyme acyl CoA cholesterol acyltransferase-1 (ACAT), which mediates synthesis of cholesteryl esters. However, we did not observe changes in the level of ACAT-1 in UBIAD1-deficient cells (Fig. 1A). Thus, reduced synthesis of cholesteryl esters in SV-589 (⌬UBIAD1) cells can be attributed to enhanced synthesis of cholesterol. In contrast, expression of SCD-associated UBIAD1 (N102S) led to the overaccumulation of neutral lipids (Fig. 4, C and D), because of the inhibition of reductase ERAD (Fig. 1A) that led to enhanced synthesis of cholesterol (Fig. 3A) and cholesteryl esters (Fig. 4B). These findings indicate that UBIAD1 (N102S)mediated inhibition of reductase ERAD significantly contributes to enhanced synthesis and accumulation of cholesterol in cultured cells. Previously, we found that all 20 SCD-associated mutants of UBIAD1 are trapped in ER membranes and confer resistance of cells to growth in medium containing SR-12813 (16), a 1,1-bisphosphonate ester that mimics sterols in acceler- Following incubation for ϳ16 h at 37°C, the cells were fixed and analyzed by immunofluorescence microscopy using IgG-1H12 (against human UBIAD1) and 4Ј6-diamino-phenylindole as described under "Experimental procedures." Images were acquired as described in the legend to Fig. 4. Scale bars, 10 m.

UBIAD1 coordinates sterol and nonsterol isoprenoid synthesis
ating reductase ERAD (21). Thus, we feel the current results with UBIAD1 (N102S) are applicable to the remaining 19 SCDassociated UBIAD1 variants. Finally, it should be noted that although UBIAD1 (N102S) blocks sterol-accelerated ERAD of reductase, sterols continued to stimulate its ubiquitination (Fig.  1D). This result is consistent with our previous hypothesis that

UBIAD1 coordinates sterol and nonsterol isoprenoid synthesis
UBIAD1 inhibits post-ubiquitination steps in ERAD of reductase (12), the molecular basis of which is currently under investigation.
In contrast to our findings summarized above, previous studies reported reduced levels of cholesterol not only in cells overexpressing wild-type, full-length UBIAD1, but also in those expressing truncated and SCD-associated variants (including N102S) of UBIAD1 (22)(23)(24). In addition, a mild accumulation of intracellular cholesterol was observed upon RNAi-mediated knockdown of UBIAD1. It should be noted that all of the previous studies utilized an indirect method employing the enzyme cholesterol oxidase to measure intracellular cholesterol levels, whereas we directly measured synthesis of cholesterol ( Figs. 2A and 3A) and cholesteryl esters (Fig. 4, A and B), as well as intracellular accumulation of neutral lipids (Fig. 4, C and  D). Direct measurement of these parameters revealed that reduced synthesis and accumulation of cholesterol/cholesteryl esters correlated with reduced levels of reductase in UBIAD1-deficient cells, whereas enhanced synthesis and accumulation of cholesterol/cholesteryl esters in cells expressing SCD-associated UBIAD1 correlated with enhanced levels of reductase (Fig. 1A).
In 1979, Faust, Brown, and Goldstein (18) found that when cells were deprived of both sterol and nonsterol isoprenoids, reductase accumulated so as to produce mevalonate that was primarily converted into cholesterol (Fig. 7A). The addition of LDL to the cells satisfied their requirements for cholesterol and partially suppressed reductase, thereby limiting synthesis of mevalonate. As a result, incorporation of mevalonate into cholesterol was reduced, but paradoxically, its incorporation into ubiquinone-10 (and presumably other nonsterol isoprenoids) was enhanced (Fig. 7B). The results of Fig. 5 show that the Golgi localization of UBIAD1 parallels synthesis of nonsterol isoprenoids as reported by Faust et al. (18). Depleting cells of sterol and nonsterol isoprenoids through incubation in LPDS and compactin triggered the accumulation of reductase (Fig. 6C),  error). B and C, SV-589 cells were set up on day 0 at 2.6 ϫ 10 4 cells/60-mm dish (for RNA isolation), 7 ϫ 10 5 cells/100-mm dish (subcellular fractionation) in medium A containing 10% FCS. On day 1, the cells were switched to medium A supplemented with 10% LPDS in the absence or presence of 10 M compactin, 0.3 g/ml 25-HC, and various concentrations of mevalonate as indicated. Following incubation for 16 h at 37°C, the cells were harvested for isolation of total RNA (B) or subcellular fractionation (C) as described under "Experimental procedures." B, total RNA from each condition was subjected to quantitative RT-PCR using primers against the indicated gene; 36B4 was used as an invariant control. Each value represents the amount of mRNA relative to that in untreated cells, which is arbitrarily defined as 1. The error bars represent Ϯ standard error of triplicate samples. HMGCR, HMG CoA reductase; FPPS, farnesyl pyrophosphate synthase. C, resulting cytosolic, nuclear, and membrane fractions were subjected to SDS-PAGE (14 -20 g total protein/lane), followed by immunoblot analysis with antibodies against HMGCS, GGPPS, SREBP-2, nucleoporin, HMG CoA reductase, SQS, UBIAD1, actin, and calnexin.

UBIAD1 coordinates sterol and nonsterol isoprenoid synthesis
which was be attributed to enhanced transcription of the reductase gene (Fig. 6B), and slowed ERAD of reductase protein (Fig.  6C). Under these conditions, a high concentration of mevalonate (10 mM) was required to stimulate the ER-to-Golgi transport of UBIAD1 (Fig. 5A). However, considerably less mevalonate (3-10-fold) caused UBIAD1 to appear in the Golgi when statin-treated cells were cultured in lipoprotein-rich FCS or LPDS plus 25-HC (Fig. 5B). Importantly, synthesis of MK-4 (used as a surrogate for production of GGpp) followed a similar pattern in that the reaction was enhanced in cells replete with sterols (Fig. 6A). These observations indicate that subcellular localization of UBIAD1 is a gauge for flux through the nonsterol branch of the mevalonate pathway.
Based on previous (12,16) and current results, we conclude that GGpp-regulated, ER-to-Golgi transport allows UBIAD1 to modulate reductase ERAD such that synthesis of nonsterol isoprenoids continues in sterol-replete cells. When cells are replete with sterols, reductase levels are suppressed by 1) reduced transcription of the reductase gene, caused by inhibition of SREBP-2, and 2) accelerated ERAD of reductase protein. However, sterols also trigger binding of UBIAD1 to a subset of reductase molecules (12). UBIAD1 binding inhibits ERAD of reductase, as indicated by reduced levels of the protein in UBIAD1-deficient cells cultured under sterol-replete conditions (Fig. 1A). This inhibition of reductase ERAD allows for production of small amounts of mevalonate that are diverted into GGpp and other nonsterol isoprenoids (i.e. dolichol, ubiquinone-10, etc.). In 1980, Brown and Goldstein (3) postulated that this diversion is facilitated by the high substrate affinity of enzymes in the nonsterol branch of the mevalonate pathway compared with that of enzymes in the sterol branch of the pathway. When GGpp accumulates to appropriate levels in ER membranes, the nonsterol isoprenoid binds to UBIAD1, causing its release from reductase. The GGpp-induced release permits transport of UBIAD1 from ER to Golgi and ERAD of reductase. The importance of this release is illustrated by the finding that GGpp-resistant UBIAD1 (N102S) is sequestered in the ER (Fig. S1) where it blocks reductase ERAD, which leads to enhanced synthesis and intracellular accumulation of cholesterol (Figs. 1, 3, and 4). Considering our recent discovery that sterol-accelerated ERAD plays a significant role in regulation of reductase and cholesterol homeostasis in whole animals (25,26), we are now poised to establish a role for UBIAD1 in reductase ERAD in vivo and determine whether UBIAD1 (N102S)mediated inhibition of the reaction contributes to the accumulation of cholesterol that characterizes SCD.

Subcellular fractionation, immunoprecipitation, and immunoblot analysis
The cells were set up for experiments on day 0 as described in the figure legends. Following incubations described in the figure legends, triplicate dishes of cells for each variable were harvested and pooled for analysis. Subcellular fractionation of cells by differential centrifugation was performed as described previously (12). For immunoprecipitations, detergent lysates were prepared and subjected to immunoprecipitation with polyclonal antibodies against the 60-kDa C-terminal domain of human reductase as described previously (21). Aliquots of detergent lysates and pellet fractions from immunoprecipitation and membrane fractions from subcellular fractionations were subjected to SDS-PAGE and immunoblot analysis. Primary antibodies used for immunoblot analysis included IgG-A9, a mouse monoclonal antibody against the catalytic domain of reductase (30); rabbit polyclonal anti-calnexin IgG (Novus Biologicals, Littleton, CO); IgG-9E10, a mouse monoclonal antibody against c-Myc purified from the culture medium of hybridoma clone 9E10 (American Type Culture Collection); mouse monoclonal anti-SOAT-1 IgG (Santa Cruz Biotechnology); IgG-P4D1, a mouse monoclonal antibody against bovine ubiquitin (Santa Cruz Biotechnology); IgG-1H12, a mouse monoclonal antibody against human UBIAD1; IgG-A6, a mouse monoclonal antibody against human cytosolic HMG CoA synthase (Santa Cruz Biotechnology); IgG-B2, a mouse monoclonal antibody against human geranylgeranyl pyrophos-UBIAD1 coordinates sterol and nonsterol isoprenoid synthesis phate synthase (Santa Cruz Biotechnology); IgG-C4, a mouse monoclonal antibody against chicken actin (BD Biosciences); IgG-53, a mouse monoclonal antibody against human nucleoporin (BD Biosciences); IgG-22D5, a rabbit polyclonal antibody against SREBP-2; and anti-SQS (Santa Cruz Biotechnology).

Immunofluorescence
SV-589, SV-589(⌬UBIAD1)/pMyc-UBIAD1 (WT), and SV-589(⌬UBIAD1)/pMyc-UBIAD1 (N102S) cells were set up for experiments on day 0 as described in the figure legends. Following incubations described in figure legends, the cells were washed with PBS and subsequently fixed and permeabilized for 15 min in methanol at Ϫ20°C. Upon blocking with 1 mg/ml BSA in PBS, coverslips were incubated for 30 min at 37°C with primary antibodies (IgG-1H12, a mouse monoclonal antibody against human UBIAD1, or IgG-9E10, a mouse monoclonal antibody against c-Myc) diluted in PBS containing 1 mg/ml BSA. Bound antibodies were visualized with goat anti-mouse IgG conjugated to Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594 (Life Technologies) as described in the figure legends. In addition, coverslips were stained for 5 min with 300 nM 4Ј,6diamidino-2-phenylindole (Life Technologies) to visualize nuclei. The coverslips were then mounted with Fluoromount G (Southern Biotech, Birmingham, AL), and fluorescence analysis was performed using a Plan-Apochromat 63ϫ/1.4 objective (Zeiss, Peabody, MA), an Axio Observer microscope (Zeiss), an), an Axiocam (Zeiss) color digital camera in black and white mode, and ImageJ software (National Institutes of Health, Bethesda, MD).

Metabolic labeling studies
Incorporation of [ 14 C]acetate and [ 3 H]mevalonate into cholesterol and that of [ 3 H]oleate into cholesteryl esters was determined as described (27,31) with minor modifications. For cholesterol synthesis experiments, the cells were set up on day 0 as described in the figure legends. On day 2, the cells received medium A supplemented with 10% FCS or LPDS and either 15 Ci/ml [ 14 C]acetate (cold acetate was added to obtain a final concentration of 0.5 mM) or 10 Ci/ml [ 3 H]mevalonate. Following incubation for 4 h at 37°C, the cell lysates were prepared, and lipids were extracted and separated by TLC on plastic-backed silica gel TLC plates (Macherey-Nagel) developed in 100% chloroform as described (32). The amount of radioactivity incorporated into cholesterol was determined by scintillation counting. To establish background radioactivity, the cells were chilled to 4°C, refed with radiolabeled medium and immediately washed and extracted. An aliquot of each sample was taken for protein determination using the BCA protein assay reagent (Thermo Scientific).
For cholesteryl ester synthesis experiments, the cell lines were cultured on day 0 as described in the figure legends. On day 1, the cells were switched to medium A containing 10% DFCS. On day 2, the cells were refed identical medium containing 0.5 Ci/ml [ 3 H]oleate and incubated for various periods of times at 37°C. The cells were subsequently washed twice with PBS ϩ 2% BSA at room temperature, followed by two washes with PBS. The lipids were then extracted with hexane/isopropanol (3:2) for 30 min at room temperature. Extracted lipids were transferred to glass tubes and mixed with recovery solution containing 0.25 Ci/ml [ 14 C]triolein plus 6.8 mg/ml cold triolein and 10 mg/ml cholesteryl ester (chloroform/methanol, 2:1 solvent). The lipids were dried down and resuspended in 40 l of heptane and spotted on TLC plates that were developed in heptane:diethlyether:glacial acid (90:30:1) solvent. The lipids were visualized by staining in iodine vapor, and incorporation of [ 3 H]oleate into CE was determined by scintillation counting. The values were corrected for background as described above; they were also corrected for recovery as judged by the percent of [ 14 C]triolein radioactivity recovered in each sample. An aliquot of each sample was taken for protein determination using the BCA protein assay reagent (Thermo Scientific Pierce).
In studies measuring incorporation of [ 3 H]menadione into MK-4, SV-589 cells were set up on day 0 as described in figure legends. On day 1, the cells were refed medium A containing 10% LPDS, 10 M compactin, and 0.2 Ci/ml [ 3 H]menadione (cold menadione was added to achieve a final concentration of 50 nM). After 16 h at 37°C, the cells were washed twice with PBS ϩ 2% BSA, followed by an additional wash with PBS. The cells were then lysed with 0.1 N NaOH; the resulting lysates were mixed with recovery solution containing 16 g/ml MK-4, 0.025 Ci/ml [ 14 C]cholesterol, and 16 g/ml unlabeled cholesterol and extracted with dicholoromethane:methanol (2:1). The lipids were dried down, resuspended in dicholoromethane, and spotted on TLC plates that were developed in chloroform. The lipids were visualized by staining in iodine vapor and incorporation of [ 3 H]menadione into MK-4 was determined by scintillation counting. The values were corrected for recovery as judged by the percent of [ 14 C]cholesterol recovered in each sample. An aliquot of each sample was taken for protein determination.

Neutral lipid staining of cells with oil red O
The cells were set up for experiments on day 0 as described in figure legends. On day 2, the cells were washed with PBS and then fixed in 4% paraformaldehyde for 20 min at room temperature. The fixed cells were then stained with oil red O solution for 10 min. Following multiple washes with PBS, the coverslips were stained with 300 nM DAPI in PBS for 5 min at room temperature. After additional washes with PBS, the coverslips were mounted with Fluoromount G. Images were acquired using a Zeiss Axio Observer Epifluorescence microscope using a 63ϫ/ 1.4 oil Plan-Apochromat objective and Zeiss Axiocam color digital camera (Zeiss, Peabody, MA) in black and white mode.

Quantitative real-time PCR
The protocol for quantitative RT-PCR was similar to that described previously (33). Total RNA was prepared from SV-589 cells using Tri-Reagent (Molecular Research Center, Cincinnati, OH). Equal amounts of RNA were treated with DNase I (DNA-free TM ; ThermoFisher Scientific). First strand cDNA was synthesized from 2 g of DNase I-treated total RNA with random hexamer primers using TaqMan reverse transcription reagents (Applied Biosystems/Roche Applied Science). Specific primers for each gene were designed using Primer Express software (Life Technologies). Triplicate RT-PCRs were set up in a final volume of 20 l containing 20 ng of UBIAD1 coordinates sterol and nonsterol isoprenoid synthesis reverse-transcribed total RNA, 167 nM forward and reverse primers, and 10 l of 2ϫ SYBR Green PCR Master Mix (Life Technologies). The relative amount of all mRNAs was calculated using the comparative threshold cycle (C T ) method. Human 36B4 mRNA were used as the invariant control. Sequences of the primers for RT-PCR used in the current study are listed in Table 1.