Expression of nonclassical HLA molecules. (a) The expression of HLA-E mRNA was evaluated in indicated OVACs by real-time quantitative PCR assay. Results are presented as fold increase in IFN-γ–treated cells (IFN) compared with untreated cells (NT). Results from one of two experiments are shown. (b) The expression of HLA-G by freshly isolated ovarian tumor cells was monitored by flow cytometry analysis. Indicated cells were stained with isotype control (mouse IgG2a) (solid line) or anti–HLA-G1 (87G) (dotted line). Results from one representative staining of at least ten are shown. (c) Western blot analysis of HLA-G expression in indicated OVACs and Caov-4 was performed using the HLA-G–specific mAb MEM-G/1 at a concentration of 1 μg/ml. The LCLs 721.221 and 721.221 transfected with HLA-G1 (721.221/G1) were used as positive and negative controls. These were diluted 1:10 because of the excess amount of HLA-G expressed by the 721.221/G1 transfectant. Cell extracts of untreated and IFN-γ–treated samples were loaded in equal amounts as determined by Western blots for β-actin (data not shown).