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. 2023 Aug 23;20(1):45–57. doi: 10.1080/15548627.2023.2247310

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© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

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Figure 3. — Increased AA uptake in HSCs results in MTOR activation, cell growth, upregulation of glycolysis and protein translation. (A-C) MTOR activity measured in HSCs from Rosa26^CreERT2 atg16l^fl/fl (as in fig. S2A) and Vav^Cre atg7^fl/fl mice. (A) Phosphorylation of MTOR (p-MTOR), (B) Phosphorylation of RPS6 (p-RPS6) and (C) Phosphorylation of EIF4EBP1 (p-EIF4EBP1) were assessed by flow cytometry. Fold change in gMFI compared with WT littermates is shown. n = 3–20 mice per group. Two-way ANOVA with post hoc Sidak’s test. (D) Relative HSC cell size was measured by forward scatter (FSC) by flow cytometry in Rosa26^CreERT2 atg16l1^fl/fl and Vav^Cre atg7^fl/fl mice. Two-way ANOVA with post hoc Sidak’s test. (E, F) Glucose uptake was assessed by fluorescent 2-NBDG using flow cytometry in HSCs from Rosa26^CreERT2 atg16l1^fl/fl mixed BM chimeras (E, n = 12) or Vav^Cre atg7^fl/fl mice (F, n_wt = 12, n_KO = 9). Data are represented as mean ± SEM with two-tailed unpaired Student’s t test. (G) Heatmap of TCA or glycolysis related genes measured by Fluidigm in HSCs from Vav^Cre atg7^fl/fl mice. Data represented as log2-fold change relative to WT. (H, I) Protein synthesis rate of HSCs was measured using the OPP-Click assay with flow cytometry in HSCs from Mx1^Cre atg5^fl/fl (H; (n_WT = 9, n_KO = 7) or Vav^Cre atg7^fl/fl (I; n = 4) mice. gMFI of OPP data are represented as mean ± SEM with two-tailed unpaired Student’s t test. WT=Cre⁻ and/or fl/⁺ littermates.