Cell lines and cell culture The human Sertoli cell line FS1 [FS1] was provided by Dr. V. Schumacher (Boston Children’s Hospital, Department of Urology; Harvard Medical School, Department of Surgery, Boston, MA, USA). The Sertoli cells were obtained from the testes of a 19 years old patient with Frazier syndrome, characterized by insufficient action of WT1 causing reduction of the downstream SOX9 expression, and with focal GCNIS. FS1 has been immortalized with both the wildtype large T-antigen and the temperature sensitive large T-antigen. FS1 was cultured in medium I, comprising Dulbecco’s modified Eagle medium (Invitrogen), supplemented with 4.5 g/l D-glucose, 40 µM L-glutamine (Sigma), 1% sodium pyruvate (Gibco, by Life Technologies), 20% fetal calf serum (PAA Laboratories GmbH), 1% nonessential amino acids, and 1% penicillin/ streptomycin (Biochrom GmbH). The human seminoma cell line TCam-2 [TCam2] was provided by Prof. Dr. H. Schorle (University Hospital Bonn, Department of Developmental Pathology, Institute of Pathology, Bonn, Germany). It was cultured in medium II, comprising Rosewell Park Memorial Institute 1640 medium (Gibco) supplemented with 2 mM L-glutamine, 10% fetal calf serum, and 1% penicillin/streptomycin). Each cell line was incubated in 75-cm2 flasks (1 x 106 cells/flask) at 37°C in a humidified incubator with 5% CO2, with medium changes every 2 or 3 days. Cells reaching 80% of confluency were used in subsequent experiments. The cell lines were authenticated by the providers and checked for mycoplasma, using a LookOut® Mycoplasma PCR Detection Kit (Sigma-Aldrich). Cell co-culture model For direct co-culture, a defined number of FS1 cells (passages 14-16) were cultured in a 6-well plate. After 24 hours, medium was replaced by a 1:1 mixture of medium I and II and a defined number of TCam-2 cells was added. For insert co-culture, FS1 (passages 14-16) (50,000 cells/well) and TCam-2 cells (50,000 cells/well) were co-cultured for 3 weeks in medium I (1.5 ml) and medium II (2 ml) using 6 well ThinCert™ cell culture inserts (Greiner Bio-One). The medium was changed on every third day. For control, FS1 cells were cultured in direct and in indirect co-culture with equine bone marrow derived mesenchymal stromal cells, eBM-MSC (kindly provided by Prof. Dr. C. Staszyk, Department of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University, Giessen, Germany). For further controls, monocultures of FS1 and TCam-2 cells were used. All control groups were cultured under the same conditions as the experimental group. For each condition, four biological replicates were generated. The co-culture experiments were repeated at least 10 times. References: [FS1] Schumacher, V.; Gueler, B.; Looijenga, L.H.; Becker, J.U.; Amann, K.; Engers, R.; Dotsch, J.; Stoop, H.; Schulz, W.; Royer-Pokora, B. Characteristics of testicular dysgenesis syndrome and decreased expression of SRY and SOX9 in Frasier syndrome. Mol Reprod Dev, 2008, 75, 1484-1494. DOI: 10.1002/mrd.20889 [TCam2] Eckert, D.; Nettersheim, D.; Heukamp, L.C.; Kitazawa, S.; Biermann, K.; Schorle, H. TCam-2 but not JKT-cells resemble seminoma in cell culture. Cell Tissue Res, 2008, 331, 529-538. DOI: 10.1007/s00441-007-0527-y
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from the cell cultures using the peqGOLD Total RNA Kit (Peqlab Biotechnologie GmbH) and purified with a RNeasy Mini Kit (Qiagen) according to the manufacture instructions.
Label
Cy3
Label protocol
After assessing RNA quality by capillary electrophoresis on a 2100 Bioanalyzer (Agilent Technologies), purified total RNA was amplified and labeled with Cy3 using a dual-color LIRAK kit (Agilent Technologies) according to manufacturer’s instructions. Briefly, the RNA samples (500 ng/ reaction) were labeled with Cy3.
Hybridization protocol
Purified Cy3-labeled antisense RNA was hybridized overnight to Agilent 60-mer oligonucleotide spotted microarray slides.Hybridization and subsequent washing and drying of the slides were performed following the Agilent hybridization protocol.
Scan protocol
The slides were scanned using a GenePix 4100A Scanner (Axon Instruments at a resolution of 2 µm/pixel.
Description
SAMPLE 3
Data processing
Image analysis was performed using GenePix Pro 5.1 software (Axon Instruments) followed by data evaluation using the R software and the Bioconductor limma package [57, 58]. The log of the mean spot signals was recorded for further analysis. Signals of replicate spots (same probes) within arrays were averaged. Intensity data were normalized using quantile normalization before calculating the average probe intensity of the array. Genes were ranked for differential expression using a moderated t-statistic.
