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. 2022 Oct 23;13:6308. doi: 10.1038/s41467-022-34064-4

Fig. 8. IL-18-enhanced and LAT2-mediated uptake of glutamine and leucine promote CD47 expression by activating the mTORC1/c-Myc axis.

Fig. 8

a Western blot analysis of HOS cells treated with PBS or IL-18 (40 ng/ml) in amino acid-sufficient medium (AA+) or medium lacking indicated amino acids for 24 h. Gln-, medium lacking glutamine; Leu-, medium lacking leucine; Gln-/Leu-, medium lacking glutamine and leucine. b Flow cytometry-based in vitro macrophage phagocytosis assay. HOS cells were treated with PBS or IL-18 (40 ng/ml) in amino acid-sufficient medium (AA+), medium deficient in leucine (Leu-), medium deficient in glutamine (Gln-), medium deficient in glutamine and leucine (Gln-/Leu-) for 24 h (n = 4 independent experiments). 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. c–e Western blot analysis of HOS cells treated with PBS or IL-18 (40 ng/ml) in amino acid-sufficient medium (AA+) or medium deficient in glutamine (Gln-, c), leucine (Leu-, d) or both glutamine and leucine (Gln-/Leu-, e) for 24 h. f Western blot analysis of HOS (left) or SJSA-1 (right) cells treated with PBS or IL-18 (40 ng/ml) in the presence or absence of rapamycin (25 nM) for 24 h. g Western blot analysis of HOS (left) or SJSA-1 (right) cells treated with PBS or IL-18 (40 ng/ml) in the presence or absence of 10058-F4 (100 μM) for 24 h. h Western blot analysis of amino acid-starved shCtrl or shLAT2 HOS cells, followed by the addition of indicated amino acids with or without IL-18 stimulation for 21 h. i Western blot analysis of shCtrl or shLAT2 HOS cells treated with PBS or IL-18 for 24 h. j Western blot analysis of HOS cells treated with PBS or IL-18 in the presence or absence of BCH, for 24 h. Data are shown as the mean ± SD. ***P < 0.001, one-way ANOVA (b). The experiment was performed three times with similar results (a, c–j). See the Source Data file for the exact P values. Source data are provided as a Source Data file.