Down-regulation of HLA-G boosted natural killer cell-mediated cytolysis in JEG-3 cells cultured in vitro  Li Li Sun, M.Sc., Yibing Han, Ph.D., Jian Hui Chen, Ph.D., Yuan Qing Yao, M.D., Ph.D.  Fertility and Sterility  Volume 90, Issue 6, Pages 2398-2405 (December 2008) DOI: 10.1016/j.fertnstert.2007.10.076 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 HLA-G gene, structure of each isoform, and RNA interference (RNAi) strategies. (A) mRNAs encoding HLA-G1 to HLA-G7 isoforms. The target sequences and primers used in reverse transcriptase–polymerase chain reaction analysis are marked. (B) The exon–intron structure of the HLA-G gene and the multiple HLA-G protein isoforms. The antibody used is able to recognize extracellular domains α1, α2, and α3. (C) The work flow of the HLA-G RNAi strategy. The selected small interfering RNA (siRNA) target sequence (19 nucleotides) spanning HLA-G exon 5 and exon 6 is underlined. The 55-mer oligonucleotides were designed and annealed to produce the siRNA template insert, which were cloned into the shuttle vector. The shuttle vector containing the correct siRNA and the adenoviral LacZ backbone plasmid were then co-transfected into HEK-293 cells. Insets A and B represent HEK-293 cells before and after transfection, respectively. In inset B, the cells have been stained with X-Gal and plaques are seen swelling or detaching from the dish. Positive cells were then harvested and the recombinant adenovirus was purified. The virus was then used to infect the JEG-3 cells; the infection rate was checked by X-Gal staining. In inset C, the JEG-3 cells with a nearly 100% infection rate are shown. HLA-G expression in the JEG-3 cells and NK cell-mediated cytotoxicity of the JEG-3 cells were checked 48 hours after infection. All photographs are taken at ×400 magnification. Fertility and Sterility 2008 90, 2398-2405DOI: (10.1016/j.fertnstert.2007.10.076) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 HLA-G expression before and after small interfering RNA (siRNA) transfection. Reverse transcriptase–polymerase chain reaction (RT-PCR) (upper panel) and Western blot (lower panel) analyses were performed using the HLA-G specific primers and the HLA-G monoclonal antibody, and β-actin was used as an internal loading control. Lane M = DNA ladder; lane 1 = uninfected cells; lane 2 = scrambled adenoviral vectors infected cells; lane 3 = siRNA adenoviral vector infected cells. The HLA-G mRNA and protein relative steady state levels (HLA-G/β-actin) were calculated and compared between the groups. All experiments were repeated three times. a,b = significant difference (P<.05). Fertility and Sterility 2008 90, 2398-2405DOI: (10.1016/j.fertnstert.2007.10.076) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 The natural killer (NK) cells-mediated cytotoxicity in HLA-G small interfering RNA transfected and control cells. (A) The raw percentages of cell cytotoxicity in the transfected and control cells using different effector cells-to-target cells (E:T) ratios. (B, C) Calculation of significant differences between the cytotoxicity percentages of the transfected and control cells. RNAi = RNA interference. All experiments were repeated three times. a,b = significant difference (P<.05). Fertility and Sterility 2008 90, 2398-2405DOI: (10.1016/j.fertnstert.2007.10.076) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions