Fig 2: Lymphocytes attach and kill trophoblasts expressing ERAP2. (A) A transfected GFP-producing JEG-3 trophoblast cell can be seen with a T cell and NK cell attached. The T cell is labeled with APC/Fire 750 anti-CD3 (dark pink) and the NK cell is labeled with PE–CD56 (yellow) and BV421 anti-CD40L (purple). Cells transfected with the empty vector (GFP+ = green) are larger and healthier and do not have T or NK cells attached. (B) Dot plots from flow cytometry showed differences in the healthy populations between cells transfected with N392 or K392 ERAP2, or an empty vector and nontransfected cells when exposed to PBMCs. Live, rounded, and healthy cells appear on the top left plot along the left vertical axis. The percentage represents the amount of gated “healthy” cells. Red cells have a varying degree of 7AAD stain. (C) The bar graph shows the percentage of apoptotic cells stained positive with 7AAD in transfected in all experimental groups compared to JEG-3 nontransfected and JEG-3 transfected with a vector without ERAP2. Cytotoxicity assays were run three times with up to three replicates of each experimental groups. The significant cell death observed in JEG-3 cells expressing N392 ERAP2 isoform (*P = 0.040). (D) The table displays the percentage of NK or T cell bound 7AAD positive N392 ERAP2 JEG-3 cells and the significant difference between the two cell types was calculated using the Student t-test (P = 0.0000213). SD, standard deviation.

Fig 3: Genetics of ERAP2 regulation. (A) LocusZoom plot of Crohn's disease and ulcerative colitis associations at the ERAP2 locus. (y-axis) −log10(P-value) of association; (x-axis) genomic location. (B) Structures of transcripts derived from each haplotype (top) and box-whisker plots (bottom three panels) between ERAP2 transcript quantitative traits (y-axis: log2(normalized gene abundance), log2(normalized isoform abundance), or isoform usage percentage)) and genotype (x-axis). (C) Correlation between ERAP2/Iso1 (orange), ERAP2/Iso3 (light blue), and ERAP2/Iso4 (dark blue) abundance (y-axis) and abundance of flu segment 4 (x-axis) segregated by rs2248374 genotype (squares indicate aa; triangles, aG; circles, GG). (D) Western blot of MoDCs before and after flu-infection from five Haplotype B homozygotes and two heterozygotes. A full-length ERAP2 protein isoform is expected at 120 kDa. At least one flu-specific ERAP2 protein isoform is expected at 49 kDa. (E) A schematic of the hypothesized regulation and function of two ERAP2 haplotypes. (N) N terminal domain; (AP) amino peptidase domain; (H) hinge domain; (C) C terminal domain; (PTC) premature termination codon.

Fig 4: Involvement of ERAP2 in viability, migration, and invasion of OSCC cells(A) SCC4 cells were transfected with control siRNA (Scr Ctrl) and ERAP2 siRNA (siERAP2), respectively. After 48 h, protein extracts (20 μg) were prepared and ERAP2 was detected with Western blot. (B) Quantitative analysis of the MTT assay. Data are presented as mean values obtained from three independent experiments. Error bars indicated the standard deviation. (C) Representative microphotographs of filters obtained from the 16 h transwell migration (D) and 24 h invasion (E) assays. Original magnification: 200×. The migration (D) and invasion (E) assays were performed using SCC4 cells in which ERAP2 was eliminated.

Fig 5: Normalized mRNA expression (copy number) of (A) endoplasmic reticulum aminopeptidase (ERAP)1 and (B) ERAP2 in placentae from first trimester (n = 12), term normotensive controls (NC; n = 12), and women with pre-eclampsia (PE; n = 12). Data are presented as median [interquartile range (IQR)]; *P < 0.05.