A short-term lung clearance assay was performed as previously described,23, 30 with some modifications to evaluate the NKG2DL-mediated immune effect in vivo. HeLa cells, which were not effectively recognized and killed by mouse NK cells, were used as internal negative controls. In brief, 36 male C57BL/6 mice were divided into three groups and were intraperitoneally injected with an IgG isotype control (300 μg per mouse; Novus, Clone 20116, MAB004), an anti-mouse NKG2D mAb (300 μg per mouse; Novus, Clone 191004, MAB1547) or an anti-NK1.1 Ab (300 μg per mouse; Biolegend, Clone PK136, 108712, San Diego, CA, USA), respectively. Twenty-four hours later, HeLa cells were labeled with PKH26 (Invitrogen, MINI26-KT, Carlsbad, CA, USA), and various BCap37 cells (transfected with miR-20a mimic, miR-20a inhibitor, or control miRNAs) were labeled with CFSE (Invitrogen, C34554). Then, the stained BCap37 cells were mixed with the stained HeLa cells of each population at a density of 5 × 106 cells in 1 ml PBS. A 0.4-ml aliquot of the cells was injected into the tail vein. Five hours later, the lungs were harvested to prepare single-cell suspensions, and flow cytometry analysis was performed. The relative ratio of tested cells to HeLa cells was calculated as the FI ratio of the tested cells to HeLa cells. Another human BC cell line (MDA-MB-231) was treated and analyzed as BCap37 cells.