Skip to main content
. 2012 Aug 3;31(17):3596–3606. doi: 10.1038/emboj.2012.218

Figure 5.

Figure 5

MEX-3C specifically regulates HLA-A but not HLA-B or HLA-C expression in primary human NK cells and modulates NK cell killing. (A) Immunoblot analysis of MEX-3C from freshly isolated and activated primary human NK cells. NK cells were purified from healthy blood donors and activated for 1–3 weeks. MEX-3C siRNA nucleofection results in efficient depletion in NK cells at 72 h post-nucleofection. (B) Immunoblot analysis of MEX-3C in the NK-92 (upper panel) and NKL cell lines (lower panel), following stimulation with IL-2 (350 U/ml) at the different time points shown. (C) Immunoblot analysis of MEX-3C downregulation in NKL cells after 72 h post-nucleofection. (D) Depletion of MEX-3C in NKL cells results in an allotype-specific increase of surface HLA-A2. Cytofluorometric analysis of HLA-A2, -B, -C, and TfR levels in mock (dashed line) and siMEX-3C cells (solid line). Isotype control (solid grey). (E) MEX-3C depletion increases HLA-A2 but not HLA-B mRNA levels in NKL cells. HLA-A2, pan-HLA-B, and MEX-3C mRNA levels were measured by qRT–PCR in mock and siMEX-3C nucleofected cells. Quantitative RT–PCR analysis of MEX-3C siRNA knockdown cells showed a significant 2.03±0.56-fold increase in HLA-A2 mRNA levels over mock cells (P-value=0.010, n=3). Transcript levels are relative to GAPDH and normalized to mock levels, set as 1 and expressed as the mean±s.d. of three independent experiments (P-value=0.01). (F) Depletion of MEX-3C increases HLA-A2 mRNA half-life. Mock (dashed line) or siMEX-3C (solid line) treated NKL cells were incubated with Actinomycin D (5 μg/ml). HLA-A2 and HLA-B mRNA levels were measured by qRT–PCR and normalized against HPRT. Results are the mean±s.d. of three independent experiments. (G) Depletion of MEX-3C in primary human NK cells results in an allotype-specific increase of surface HLA-A (HLA-A2 and -A3) but not HLA-B or -C. Cytofluorometric analysis of HLA-A2, -A3, -B, and -C levels in mock (dashed line) and siMEX-3C nucleofected cells (solid line). Isotype control (solid grey). (H) Reduced target cell killing in MEX-3C depleted primary NK cells. 51Cr release assay (percent specific lysis) using mock siRNA (dashed line) or siMEX-3C (solid line) nucleofected NK cells against K562 target cells. Error bars show the standard error of the six replicates at each point. The results are representative of three independent experiments using polyclonal NK cells from unrelated donors.