NOEY2 expression in cells and tissues. (A) Northern blot analysis of OSE cells (right three lanes) and ovarian cancer cell lines (remaining lanes). (B)Northern blot analysis of normal breast epithelial (NBE) cells (right four lanes) and breast cancer cell lines (remaining lanes). (C) Northern blot analysis of OSE cells (left three lanes) and ovarian cancer cells purified from patients’ ascites fluid (remaining lanes). (D) Northern blot of multiple human tissues. Poly(A)⁺ RNA (2 μg) from multiple human tissues was supplied in a blot prepared by CLONTECH. (E) Western blot analysis of positive control (NIH 3T3 transiently transfected with NOEY2 cDNA), OSE cells (left lanes 2–4), and ovarian cancer cell lines (remaining lanes).

Metaphase mapping of NOEY2 by fluorescence in situ hybridization. A P1 clone containing NOEY2 was mapped to 4′,6-diamidino-2-phenylindole-banded human chromosome 1p31 in normal human lymphocytes. (Inset) Localization of NOEY2 (green) on chromosome 1 counterstained with propidium iodide and 4′,6-diamidino-2-phenylindole compared with 4′,6-diamidino-2-phenylindole-banded chromosome 1.

Comparison of NOEY2 with Ras and Rap family members. The four conserved GDP/GTP binding domains and the CAAX motif are underlined. Bold type indicates residues conserved in nearly all GTPases. Amino acids are designated according to the single letter code.

NOEY2-induced growth inhibition of ovarian and breast cancer cell lines. (A) Colony formation after NOEY2 cDNA transfection. NOEY2 in the sense orientation inhibited growth of OVCA433, OVCA429, Hey, and SKBr3 (P < 0.001). (B) Inhibition of cyclin D1 promoter activity in Saos-2, NIH 3T3, SKBr3, and Hey cells. Luciferase activity in cells transfected with the sense construct was expressed as a percentage of the activity in cells transfected with the antisense vector. The results are from representative experiments performed in triplicate.

Monoallelic expression and imprinting of NOEY2. (A) Monoallelic expression. Shown is analysis of coding +231 G/A allele expression by using HhaI digestion of a reverse transcription–PCR transcript (lanes 2, 4, 6, 8) and genomic DNA as controls (lanes 1, 3, 5, 7) from homozygote G allele in OSE001 and heterozygote G/A alleles in BT20, CAOv3, and OSE 031. Arrowheads: a, 326 bp for an HhaI-uncut fragment of A allele; b, 206 bp; and c, 120 bp for HhaI-cut two fragments of G allele. (B) Maternal imprinting of NOEY2. Genotype of TA repeat length polymorphism of three families (F1, F2, and F3) was shown as peaks. Maternal (lane 1); paternal (lane 2); offspring from the same families (lanes 3 and 5; D, daughter; S, son). Maternal origin of methylated allele is demonstrated in each offspring (lanes 4 and 6). (C) DNA fragments analysis of LOH and methylated retained allele in ovarian cancer patients. Genotype of TA repeat length polymorphism of normal DNA from two ovarian cancer patients (lane 1): one allele is lost in tumor DNA (lane 2); the retained allele is methylated (lane 3). (D) The genomic structure of NOEY2 and the location of the primers and polymorphisms.