Role of BAG6 for immune detection in vitro and in an in vivo xenograft model. (A) Western blot (WB) of 293T cell lysates to verify BAG6 downregulation in BAG6-shRNA–transduced 293T cells (k.d.) in comparison with wt and control shRNA-transduced cells (ctrl). Actin was used as a loading control. Exosomes derived from these cell lines were checked for exosomal markers HSP70 and CD9. BAG6 knockdown in exosomes was demonstrated via flow cytometry (supplementary Figure 2). (B) The wt, control shRNA (ctrl), and BAG6 shRNA (k.d.) transduced 293T cells were used as target cells in an NK cell cytotoxicity assay (1 representative experiment of 3). (C) The wt, ctrl, and k.d. 293T cells were implanted subcutaneously (3 × 106 cells) into immunodeficient SCID/beige mice. The treatment groups received human lymphocytes (1 day after tumor cell transplantation, 3 × 106 PBMC intraperitoneally.). The tumor incidence was monitored (left: no PBMCs; right: with PBMCs; n = 9 or 10 for ctrl and k.d.; n = 5 for wt in both settings).
