Inhibition of ERK prevents the ability of NK cells to form NKIS. (A) NKIS were visualized by fluorescence microscopy. Erythroleukaemia K562 cells were mixed with activated NK cells (ratio NK/K562, 2:1) and cultured for 20 minutes at 37°C on a poly-lysine–coated slide and stained with anti-phospho-tyrosine (4G10, green stain) and F-actin (red stain). Nuclei were stained with DAPI (blue stain). In the differential interference contrast micrograph (DIC) on the left (63×), a human NK cell is shown conjugated to a K562 cell and the synapse is indicated with an arrow. One representative experiment, of at least 3 independent ones, is shown. Signal intensity for 4G10 and F-actin was quantified with ImageJ software. (B) NK cells were cultured with K562 for 20 minutes at 37°C (ratio NK/K562, 2:1) and PI3K (p85) and pERK protein expression was evaluated by Western blot analysis. GAPDH was used as loading control. (C) Degranulation ability by NK cells cultured with IPS-MSCs or H9-MSCs for 4 days was evaluated using the CD107a assay. NK cells were stained with anti-CD56–APC and with anti-CD107a–PE and analyzed by flow cytometry. Monensin (10μM) was added to prevent the acidification of the endosomal compartment and the degradation of the granules secretion. The percentages listed represent the ratios of CD56⁺/CD107a⁺ cells. Results are representative of 4 independent experiments. Results are representative of 4 independent experiments and are shown as the means ± SD of triplicate samples.