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cDNA representing the rat GnRH receptor was cloned into pcDNA3 as described and validated previously [ 22 23 ] . cDNAs representing RGS3 and RGS3T (RGS3 amino acids 314-519) were cloned into the eukaryotic expression vector, rcCMV, as described [ 24 ] . COS-1 cells cultured as detailed before [ 23 ] were co-transfected with GnRH receptor cDNA and RGS3 or RGS3T cDNA using Lipofectamine (Life Technologies, Gaithersburg, MD) [ 23 ] , and 72 h later, treated with 10 -7M GnRH-A for 0, 10, 20, or 30 sec before being collected for IP 3 measurements [ 22 23 ] .
Human prostate carcinoma cells, LNCaP (ATCC, Rockville, MD), were maintained and propagated in T25 flasks in RPMI 1640 media (GIBCO-BRL, Gaithersburg, MD) supplemented with 10% FCS (Gemini Bio-Products, Calabasas, CA), 2 mM glutamine, Fungizone (1 μg/ml) and penicillin (200 unit/ml)/ streptomycin (200 μg/ml) (referred as complete medium) in a 37°C humidified 5% CO 2 incubator.
Apoptotic activities in TM10 and TM2H cells in the absence and presence of 2.5 μM SAHA were examined by two procedures: the TACS 2TdT In Situ Apoptosis Detection assay following the manufacturer's instructions (Trevingen, Gaithersburg, MD, USA), and the DNA Fragmentation Assay described elsewhere [ 29].
Total RNA was extracted from human breast cell lines, frozen breast tumor tissues and normal breast tissues with Trizol reagent (Invitrogen, Gaithersburg, MD, USA) in accordance with the manufacturer's protocol.
Trizol™ reagent (Life Technologies, Gaithersburg, MD) was used to prepare total RNA from fetal and adult mouse tissues.
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