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. 2015 Apr 7;7(6):714–734. doi: 10.15252/emmm.201404571

Figure 2.

Figure 2

Co-incubation of B16F10 melanoma with platelets results in Asm secretion and activation and ceramide release to mediate metastasis
  • A Platelets were isolated from wild-type (WT) or Asm-deficient (Asm−/−) mice by density-gradient centrifugation, and 1 × 107 platelets were incubated with 1 × 105 B16F10 cells. The samples were lysed, and Asm activity was determined. In unstimulated samples (time point of co-incubation 0 s), tumor cells and platelets were admixed after lysis.
  • B The formation of ceramide was determined by a diacylglycerol (DAG) kinase assay (upper panel) and mass spectrometry (lower panel). In unstimulated samples (time point of co-incubation 0 s), tumor cells and platelets were admixed after lysis.
  • C, D For determination of the secretion of Asm by platelets into the supernatants (C), tumor cells and wild-type (WT) or Asm-deficient (Asm−/−) platelets were co-incubated for 20 s, the samples were pelleted, the supernatants were removed and acidified, and the Asm activity was measured in the presence or absence of Zn2+. For measurement of surface Asm and ceramide, samples were co-incubated as indicated (C, D) and pelleted; the supernatants were discarded, incubated with anti-Asm antibodies, washed, and lysed; and Asm immunocomplexes were immobilized and subjected to immunocomplex enzyme assays. Surface ceramide was measured by incubation of intact cells with DAG kinase in the presence of [32P]γATP followed by extraction and measurement of [32P]-ceramide. Omission of one cell type indicated by “−” here and thereafter.
  • E Treatment of B16F10 tumor cells for 2 min with 1 U/ml ASM or 10 μM C16 ceramide restores in vivo metastasis in Asm-deficient mice. After treatment, the cells were injected intravenously into Asm-deficient (Asm−/−) mice. Controls were left untreated prior to injection. The number of metastases was determined 14 days after tumor cell injection.
Data information: Displayed is the mean ± SD of 4 (A–D) or 9 (E) experiments. Statistical significance was determined by analysis of variance (ANOVA) followed by a Tukey's multiple comparisons test. P-values are given.