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. 2019 Mar 29;10:1444. doi: 10.1038/s41467-019-09431-3

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — mTOR and ERK inhibition phenocopy the expansion defect of Shp-2-deficient NK cells. a Splenocytes from C57BL/6 wild-type mice were cultured in vitro for 30 min in the presence of 50 ng/ml IL-15 (blue) and in the presence of 50 µM PD98059 or 250 nM Torin2, or left untreated (white). Phosphorylation of the indicated proteins was measured by flow cytometry. b Splenocytes from Ncr1^Ki Ptpn11^wt/wt and Ncr1^Ki Ptpn11^fl/fl mice were cultured in vitro for 30 min in the presence of 50 ng/ml IL-15 (blue and red, respectively) or left untreated (white and dark gray, respectively). Phosphorylation of Akt (position 308) was measured by flow cytometry in CD27 SP, DP, and CD11b SP NK cell subsets (gated as NK1.1⁺ and CD3/CD19⁻). For quantification, the average of the untreated NK cells from Ncr1^Ki Ptpn11^wt/wt mice was set as 100%. c, d NK cells from C57BL/6 wild-type mice were cultured in vitro with 50 ng/ml IL-15 for 4 days; PD98059 was added daily (day 0, 1, 2, and 3) at a concentration of 25 μM, while Torin2 was added once (day 0) at a concentration of 5 nM. c Histograms represent the amount and cell division (CTV dilution) of living NK cells in each condition (quantitative flow cytometry acquisition) and graph the number of living NK cells (gated as propidium iodide (PI)^–NK1.1⁺CD3/CD19^–) after 4 days of culture for each condition. d Graphs depict the geometric mean of FSC and SSC of non-divided NK cells across conditions (gated as PI^–NK1.1⁺CD3/CD19⁻ and cell trace violet (CTV) high). For quantification, the average of IL-15-expanded NK cells was set as 100%. Results represent the mean ± SEM of n = 9 (a), n = 9 (Ncr1^Ki Ptpn11^wt/wt) and n = 10 mice (Ncr1^Ki Ptpn11^fl/fl) (b) and are a pool of two independent experiments (a, b). Results represent the mean ± SD of n = 5 technical replicates and are representative of two independent experiments (c, d). Statistical comparisons are shown (a–d); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, NS, non-significant; Student’s t-test. Source data are provided as a Source Data file