Fig. 7: Amino acid transporter LAT2-mediated amino acid uptake is necessary for IL-18-induced CD47 expression in osteosarcoma cells.

a Quantitative real-time PCR analysis of the indicated genes in HOS cells treated with IL-18 (40 ng/ml) or PBS for 24 h (n = 3 independent experiments). b Western blot analysis of LAT2 expression in HOS cells following IL-18 or PBS treatment for 24 h. c Western blot analysis of CD47 and LAT2 expression in shCtrl or shLAT2 HOS cells. d Western blot analysis of shCtrl or shLAT2 HOS cells treated with PBS or IL-18 for 24 h. e Western blot analysis of HOS cells treated with PBS or IL-18 in the presence or absence of BCH, for 24 h. f Flow cytometry-based analysis of macrophage phagocytosis of shCtrl HOS cells or shLAT2 HOS cells after treated as indicated. Macrophages were defined as F4/80⁺ (labeled with PE) events, and tumor cells as GFP⁺ events. F4/80⁺, GFP⁺ events represented macrophages that had phagocytosed tumor cells (n = 3 independent experiments). g Immunohistochemistry analysis of LAT2 expression (LAT2 H-score) in matched osteosarcoma specimens from the same patient before and after chemotherapy (n = 81 patients). h Kaplan–Meier curves of overall survival (left) and disease-free survival (right) of osteosarcoma patients stratified by ΔLAT2 expression. High ΔLAT2 expression (ΔLAT2^high): ΔLAT2 H-score > 0.45; low ΔLAT2 expression (ΔLAT2^low): ΔLAT2 H-score < 0.45. ΔLAT2 H-score for each patient was obtained from subtracting LAT2 H-score of pre-chemotherapy specimen from that of paired post-chemotherapy specimen. i Heatmap showing the relative concentrations of amino acids in shCtrl or shLAT2 HOS cells stimulated with PBS or IL-18 (n = 3 independent experiments). j Western blot analysis of amino acid-starved HOS cells treated with IL-18 or PBS in amino acid-free (AA−) or amino acid-sufficient (AA+) medium for 21 h. Data are shown as the mean ± SD. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed Student t test (a), one-way ANOVA (f), paired two-tailed Student t test (g) or log-rank test (h). The experiment was performed three times with similar results (b–e, j). See the Source Data file for the exact P values. Source data are provided as a Source Data file.