Extended Data Fig. 6: The phenotype of NK cells after inhibition of sphingomyelin synthesis.

a, CLSM showing the binding of human liver-cancer cells and NK cells. NK cells freshly purified from normal donor peripheral blood and were co-cultured with HepG2 (liver cancer cell line) cells. Peripheral NK cells were treated with or without the small molecule sphingomyelin synthase inhibitor D609 (25, 100, 400 μM) or PBS (control) for 24 h. The large cells on the left are HepG2 cells; the small cells on the right are NK cells. Green, F-actin staining. Scale bar, 0.5 μm. b–c, Flow cytometry showing apoptosis (annexin-V⁺) in NK cells after treatment with D609 (25, 100, 400 μM) or PBS (control) for 24 h or knockdown of sphingomyelin synthase 1 (si-SGMS1). n = 5 samples per group. c (left), Western blotting analysis of SGMS1 expression in primary NK cells isolated from normal donor peripheral blood. The siRNA-SGMS1 and siRNA-mock were transfected into peripheral NK cells for 24 h until analysis. d, Flow cytometry assaying proliferation (Ki67⁺) in NK cells after treatment with D609 (25, 100, 400 μM) or PBS (control) for 24 h or knockdown of sphingomyelin synthase 1 (si-SGMS1). n = 5 samples per group. e–f, Flow cytometry assay of the percentage of IFN-γ, perforin, and granzyme-B (GZMB) producing cells among IL-12+IL-18 or PBS (Control) stimulated NK cells from normal donor peripheral blood. The MFI of IFN-γ in IL-12+IL-18-stimulated IFN-γ⁺ NK cells, perforin in IL-12+IL-18-stimulated perforin⁺ NK cells, and GZMB in IL-12+IL-18-stimulated GZMB⁺ NK cells are presented as the mean ± s.d.. Peripheral NK cells pre-treated with D609 (25, 100, 400 μM) or PBS (control) for 24 h before IL-12 and IL-18 stimulation. n = 5 samples per group. N.D., not detected. g, FACS assay of CD107a levels in peripheral NK cells. Peripheral NK cells were treated with D609 or PBS for 24 h. Then, NK cells were co-cultured (Co-cultured group) with or without (Control, single-cultured group) HepG cells before FACS assay. n = 5 donor samples per group. h, i (related to Fig. 5m and n) HepG2 cells and (J) K562 cells were used as target cells in cytotoxicity assays with indicated NK cells. % cytolysis is shown (n = 5 per group). h, j, Purified NK cells were co-cultured with HepG2 cells or K562 cells for cytotoxicity assays after treatment with the small molecule sphingomyelin synthase inhibitor D609 (25, 100, 400 μM) or PBS (control) for 24 h. i, Purified NK cells were co-cultured with HepG2 cells for cytotoxicity assays after knockdown of sphingomyelin synthase 1 (si-SGMS1) or control (si-mock) for 24 h. j, n = 3 per group. k, The ADCC (antibody-dependent cell-mediated cytotoxicity) activity assays of NK cells. Target cells, HepG2 cells (An EpCAM+ liver cancer cell line); Ab, humanized antibodies against EpCAM; peripheral NK cells pre-treated with D609 (25, 100, 400 μM) or PBS (control) for 24 h. n = 3 samples. l, SEM images showing the membrane protrusions of purified NK cells. Representative SEM imaging (left) and the mean number of membrane protrusions (right) in each sample are shown. NK cells were treated with IL-2, IL-2 + IL-15, or IL-2 + IL-15 + IL-12. Each dot represents one sample. n = 6 in each group. Scale bar, 1 μm. m, Fresh NK cells isolated from liver cancer patients (tumor and liver tissue outside tumor) and normal donor peripheral blood (n = 8 in each group). Peripheral NK cells were treated with or without the small molecule sphingomyelin synthase inhibitor D609 (25, 100, 400 μM) or PBS (control) for 24 h (n = 5 in each group). Using a Transwell™ apparatus, we tested the migration of the NK cells from the upper compartment to the lower compartment. Counting the numbers of NK cell migrating to the lower layer. n, Flow cytometry assay of CD29 and CD18 levels in intratumoral NK cells, liver NK cells and peripheral NK cells. n = 5 samples per group. Data are the mean ± s.d. Data were analysed by two-way ANOVA.

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