Fig. 3

mTORC1, but not Akt, is required for cMyc expression in IL-2/IL-12-stimulated NK cells. a NK cells were left unstimulated (US) or stimulated with IL-2/IL-12 for 18 h and Akti-1/2 (2 μM) was added for the last 1 h of activation, before immunoblot analysis for levels of cMyc, phosphorylated Akt on serine 473 (pAkt) and β-actin. b NK cells were stimulated with IL-2/IL-12 in the presence or absence of Akti-1/2 (2 μM) for 18 h before immunoblot analysis of cMyc and S6 ribosomal protein (S6, loading control) levels. c–g NK cells were left unstimulated or stimulated with IL-2/IL-12 in the presence or absence of Akti-1/2 (2 μM) for 18 h, then analysed by flow cytometry for FSC-A and CD71 expression (c), IFNγ production (d, e) and granzyme B expression (f, g). h NK cells were left unstimulated or stimulated with IL-2/IL-12 for 18 h and Akti-1/2 (2 μM) or rapamycin (20 nM) were added for the last hour of activation as indicated. Samples were subjected to immunoblot analysis for levels of phosphorylated S6 ribosomal protein on serine 235/6 (pS6), phosphorylated p70 S6 kinase on serine 389 (pS6K) and S6 ribosomal protein (S6). i, j NK cells were stimulated with IL-2/IL-12 for 30 min, 60 min (i) or 18 h (j) in the presence or absence of rapamycin (20 nM) as indicated before immunoblot analysis for cMyc and β-actin expression. Data are mean ± s.e.m of 6 experiments (e, g), or representative or 3 (a–j) or 6 (c–f) individual experiments. Statistical analysis was performed using Student's t-test (e) or a one-sample t-test vs. a theoretical value of 1 (g); ns non-significant