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. 2018 Nov 8;9:4685. doi: 10.1038/s41467-018-07024-0

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© The Author(s) 2018

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. 3 — Activation of the Akt/mTOR pathway by reverse mKitL signaling. a Intracellular flow cytometry analysis of Akt S473, mTOR S2448, and Rps6 S235/S236 phosphorylation in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells as indicated (N = 7, two experiments). Values are normalized MFI (NIH-Venus mean = 100). Bars show mean ± s.e.m. P-value is shown (Student’s t-test). b Representative intracellular flow cytometry plots measuring Rps6 S235/S236 phosphorylation in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells, as indicated. Gating shows cells with high phospho-Rps6 levels. Data are representative of seven experiments. c Percentage of phospho-Rps6 positive cells in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells in b (N = 7, two experiments). Bars show mean percentage of S235/S236 positive cells ± s.e.m normalized to the average value for NIH-Venus ( = 100). P-value is shown (Student’s t-test). d CREB phopho-Ser133 MFI (left) and percentage CREB phopho-Ser133 positive cells (right) in NIH3T3 cells co-cultured with NIH-Venus or NIH-Kit cells, as indicated (N = 8, two experiments). Bars show mean ± s.e.m. P-value is shown (Student’s t-test). e Representative intracellular flow cytometry plots measuring Ki67 in NIH3T3 cells co-cultured with NIH-Kit cells and treated for 9 h with DMSO, Akt inhibitor (Akti1/2) or CREB inhibitor (666–15), as indicated. Gating shows the Ki67^high population. f Percentage of Ki67^high cells (left) and Ki67 MFI (right) in NIH3T3 cells co-cultured with NIH-Kit cells and treated for 3, 6, or 9 h (as indicated) with DMSO (n = 13, two experiments), Akt inhibitor (Akti1/2), or CREB inhibitor (666–15), as indicated (N = 6, two experiments, except the Akti1/2 3 h time point where N = 9, two experiments). Bars show mean ± s.e.m. P-value is shown (Student’s t-test)