221, an Epstein–Barr virus-transformed B-cell line, and K562, an erythroleukemic line, were cultured in RPMI 1640 with 10% FBS. Human pNKs were enriched from fresh blood using a RosetteSep® Human NK Cell Enrichment kit (Stemcell Technologies, Vancouver, British Columbia, Canada) as per the manufacturer’s protocol. The purity of the isolates was assessed by flow cytometery using CD56 surface marker. Freshly isolated cells were cultured in RPMI 1640 (Gibco) with 15% FBS supplemented with 500U/mL IL2 (R&D systems) and irradiated RPMI 8866 cells and allogeneic Ku-0059436 ic50 peripheral blood mononuclear cells (PBMCs).
Primary Abs used for this study were rabbit polyclonal anti-IQGAP1 (sc-10792 Santa Cruz Biotechnology, CA, USA), mouse monoclonal anti-perforin (MAB4616, clone, Chemicon, Temecula, CA, USA), anti-IQGAP1 mAb (05–504, Upstate Biotechnologies). The secondary Abs were Alexa fluor 546-conjugated goat anti-rabbit IgG, Alexa Fluor 594-conjugated goat anti-rabbit IgG, Alexa Fluor 350-conjugated goat anti-mouse IgG, Fluor 405-conjugated goat anti-mouse IgG, and the phalloidin conjugates with Alexa fluor 488 or Alexa fluor
Torin 1 manufacturer 546 were all purchased from Molecular Probes (Eugene, OR, USA). The effector target conjugates were generated as described previously 39. Conjugates containing effector and target cells were stained for actin, IQGAP1, and perforin. Maturation stages of the NKIS were categorized into three broad categories based on the localization of perforin-containing granules in the NK cells relative to the synapse. Immature NKISs were defined as those where a contact between the NK and the target had been established but the granules had not been mobilized toward the
contact sites. Mature NKISs were those in which granules were accumulated and aligned at the interface between the effector and the target cell. Mid-synapses were categorized as those that showed partial polarization of the granules toward the contact sites. Imaging was performed using 63X on a Zeiss LSM 710 Observer station. Image analysis was done on AxioVision software version 4.8.1. In order to assess the translocation of the MTOC toward the NKIS, conjugates containing effector and target cells were stained for actin, 6-phosphogluconolactonase IQGAP1, α-tubulin, and perforin. The location of MTOC was determined and the distance was measured from the centroid of the α-tubulin defined MTOC to the NKIS, using distance measurement tool of Axiovision 4.8.1 software. Two TRC clones in the pKLO1 vector (Openbiosystems, Huntsville, AL, USA) RHS3979-9614686 (designated construct 1) and RHS3979-9614684 (designated construct 2) were used to separately silence IQGAP1 expression. Viral packaging and the generation of YTS transductants expressing these clones were performed according to the previously described methods 39. Cell-mediated cytotoxicity was measured using a chemiluminiscence-based assay, CytoTox-Glo™, as per the manufacturer’s protocol (Promega cat no. G9291). Effector cells (i.e.