Fig. 4

MITF promotes NKG2D ligand shedding in melanoma cells. a ELISA assay in SK-MEL-28 cells to determine the concentration of MICA in the extracellular medium. The data show the mean ± SD and the variation in the concentration of MICA after irradiation (IR, 10 Gy) was statistically significant with respect to the control at all times tested (p < 0.05). b ELISA assay in SK-MEL-28 cells to determine the concentration of MICB in the extracellular medium (*p < 0.05; ns, no significant) MICB was determined at the same time (24 h) in control and irradiated (IR, 10 Gy) cells. c Flow cytometry determination of Rae1δ in B16/F10 and effects of MITF depletion. Cells were non-irradiated or irradiated (IR, 10 Gy, 24 h). Histogram represents MFI (Mean Fluorescence Intensity) in assayed conditions. *p < 0.05 when compared with their respective non-irradiated cells. **p < 0.005 when comparing both irradiated cells. d Athymic (Fox1nu) and C57BL/6 mice were immunized by injection in the tail vein with ex vivo irradiated B16/F10 cells (10 Gy; 24 h). One week after the injection, animal were sacrificed and NK cells were purified from their spleens. Purified NKs were co-cultured with YAC-1 cells, irradiated B16/F10 or irradiated B16/F10-MITF-KO. NK cytotoxicity was evaluated by a ⁵¹Cr-release assay at two effector/target ratios (E/T). The results are representative of three independent experiments (*p < 0.05)