transiently transfected with two siRNAs, i.e., siRNA1 (sense: GCUUCCAGUUCAGAUGCCATT; and anti-sense: UGG- CAUCUGAACUGGAAGCTT) and siRNA2 (sense: GGAA- CUAACCAAAAUUCAATT; and anti-sense: UUGAAUUUUG- GUUAGUUCCTC) or with non-targeting control RNA (4611G) (final concentration 20 nM), using Lipofectamine RNAiMAX (Life Technologies). Relative amount of endogenous Mamld1 mRNA against B2m (b2-microglobulin) was determined by the TaqMan real-time PCR method using the probe-primer mix on ABI PRISM 7000 (Life Technologies) (Assay No.: Mm01293665_m1 for Mamld1; and Mm00437762_m1 for B2m). Steroid metabolite measurements MLTCs are known to have the capacity to produce T primarily via D4-pathway, although the amount of T production remains small primarily because of low 17a-hydroxylase and Hsd17b3 activities [6]. MLTCs are also known to retain responsiveness to human chorionic gonadotropin (hCG) [6–8]. Thus, after 48 hours of incubation of transfected MLTCs in 12-well plates with 1 ml of culture medium (an initial cell count: 16105 cells/well), hCG (Mochida Pharmaceutical) was added to the media at a final concentration of 50 IU/L, and the culture media were obtained at one hour after the addition of hCG. Subsequently, steroid metabolites in the T production pathway were measured by the liquid chromatography-tandem mass spectrometry (ASKA Pharma Medical). This experiment was performed three times. Gene expression analyses Real-time reverse transcriptase (RT)-PCR and microarray analyses were performed using total RNA extracted from MLTCs that were harvested at the time of steroid metabolite measure- ments. For real-time RT-PCR analysis, 1 mg of total RNA was examined for relative mRNA dosage against B2m by the TaqMan Gene Expression Assay on ABI PRISM 7000 (Assay No.: Mm00446826_m l for Nr5a1 (Sf1); Mm00441558_m1 for Star; Mm00490735_m1 for Cyp11a1; Mm01261921_mH for Hsd3b1; and Mm00484040_m1 for Cyp17a1). In addition to the genes for steroidogenic enzymes involved in T biosynthesis, we also studied Insl3 (Mm01340353_m1) for gubernacular development that is expressed in Leydig cells [9,10]. This experiment was repeated three times. For microarray analysis, 300 ng of total RNA was converted into cRNA associated with Cyanine-3 labeled CTP using RNA Spike-In Kit and Quick Amp Labeling Kit, and was subjected to hybridization on Whole Mouse Genome Oligo Microarray in triplicate (4644 K G4122F) (Agilent Technolo- gies). Subsequently, fluorescent signals were detected by Agilent Scanner, and were analyzed by GeneSpring GX10 (Tomy Digital Biology). The microarray data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession number GSE26913 (http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE26913). All data is MIAME compliant and the raw data has been deposited in a MIAME compliant database (GEO), as detailed on the MGED Society website (http://www.mged.org/Workgroups/MIAME/miame. html). Cell proliferation assays The number of viable MLTCs transfected with two siRNAs or with non-targeting RNA was calculated by the colorimetric method [11,12], using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). The detailed procedure has been described in the manufacturer’s protocol. In brief, MLTCs were cultured in 96-well plates (an initial cell count: 16104 cells/well), and the cell number was determined every 24 hours by measuring Mamld1 Knockdown in Mouse Leydig Tumor Cells the absorbance on a plate reader (Molecular Device) at 490 nm. This method is based on a positive correlation between the number of viable cells and the absorbance until the cells become confluent, and our preliminary studies showed a good correlation until the absorbance of ,2.0 (,66105 cells/well) (Figure S1). This experiment was performed three times. Statistical analysis Statistical significance was examined by Student’s t-test or by Mann-Whitney’s U-test. P,0.05 was considered significant. Results Steroid metabolite measurements The mean steroid metabolite concentrations are shown in Figure 1, together with the mean endogenous Mamld1 mRNA levels that were markedly reduced in both siRNA1- and siRNA2- transfected MLTCs at the time of steroid metabolite measure- ments. The concentrations of pregnenolone and progesterone remained comparable between the culture media with siRNA- transfected MLTCs and those with non-targeted MLTCs, whereas the concentrations of 17-OH pregnenolone, 17-OH progesterone, dehydroepiandrosterone, androstenedione, and T were signifi- cantly lower in the culture media with siRNA-transfected MLTCs than in those with non-targeted MLTCs. Furthermore, compar- ison of the steroid metabolite concentrations in the media with non-targeted MLTCs confirmed revealed the D4-pathway dom- inant T production, markedly low 17a-hydroxylase activity and well preserved 17/20 lyase activity for both D4- and D5-pathways, and extremely low Hsd17b3 activity in MLTCs. These results indicated that Mamld1 knockdown further reduced 17a-hydroxy- lase activity that was originally low in MLTCs. Gene expression analyses Real-time RT-PCR and microarray analyses showed signifi- cantly decreased Cyp17a1 expression (,70%) in both siRNA1- and siRNA2-transfected MLTCs (Figure 2). Although Cyp11a1 and Hsd3b1 expressions were found to be reduced in siRNA1- transfected MLTCs by real-time RT-PCR and microarray analyses respectively, such reduced activities were not reproduced in siRNA2-transfected MLTCs. The siRNAs knockdown did not affect the expressions of Nr5a1 (Sf1), Star, Por, and Insl3. The assessment of Hsb17b3 was impossible, because of its extremely low expression. In addition, 47 genes including a Notch-related gene Hey1 were significantly up-regulated (fold change .2.06) and 38 genes were significantly down-regulated (fold change ,0.56) in both siRNA1- and siRNA2-transfected MLTCs (Table S1 and Table S2). However, Mamld1 knockdown had no discernible effect on the Hes3 expression level (siRNA1: fold change 0.92, P = 0.80; siRNA2: fold change 1.43, P = 0.35). The microarray data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession number GSE26913 (http://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE26913). Cell proliferation assays The results are shown in Figure 3. The mean endogenous Mamld1 mRNA levels were sufficiently suppressed for 120 hours in both siRNA1- and siRNA2-transfected MLTCs. Under this condition, the absorbance values for the siRNA-targeted and non-targeted MLTCs showed a roughly linear increase until 72 hours (absorbance ,2.0). In this linear proliferative phase, although the absorbance values were significantly decreased in siRNA2-treated MLTCs at 24 and 48 hours after the transfection, PLoS ONE | www.plosone.org 2 April 2011 | Volume 6 | Issue 4 | e19123