Figure 2.
Impact of constitutive AKT activation on HSC function in obesity. (A) Gene set enrichment analysis (GSEA) for upregulated genes in db HSC-SLAMs. Numbers in parentheses show the number of genes in indicated gene sets. (B) Scheme of manually curated upregulated (blue) and downregulated (green) genes in HSC-SLAMs identified by genome-wide gene expression analysis and linked to the phosphatidylinositol pathway and AKT signaling. (C) Representative fluorescence-activated cell sorting (FACS) plot and histogram (left panel) showing the gating strategy and levels of AKT and STAT3 phosphorylation in the HSC-SLAM compartment isolated from the BM of 4-month-old control and db mice. Right panel shows relative average mean fluorescence intensity (MFI) ± standard deviation (SD) of AKT, STAT3, STAT5, and pERK phosphorylation in db HSCs compared with control (n = 5). Student t test; *P ≤ .05. (D) Experimental scheme for in vivo pharmacologic AKT inhibition (left panel). Right panel shows relative average MFI ± SD of AKT phosphorylation in each experimental group (n = 3). Two-way ANOVA with Tukey’s post hoc test; *P ≤ .05; **P ≤ .01. (E) Mean percentage ± SD of HSC-SLAMs and MPPs (LSK CD48+) in the BM of 4-month-old control and db mice treated with vehicle (veh) or the AKT inhibitor MK-2206 for 2 weeks (n = 12-20 from 5 independent cohorts). Two-way ANOVA with Tukey’s post hoc test; ****P ≤ .0001. (F) Representative FACS histogram (left panel) and average MFI ± SD (right panel) showing levels of CD34 marker on HSC-SLAMs isolated from 4-month-old control and db mice treated with vehicle or the AKT inhibitor MK-2206 for 2 weeks (n = 11-12 from 5 independent cohorts). Two-way ANOVA with Tukey’s post hoc test; ***P ≤ .0005. (G) PB chimerism in competitive reconstitution assays using purified HSC-SLAMs isolated from the BM of control and db mice treated with vehicle or the AKT inhibitor MK-2206 for 2 weeks. The right graph shows myeloid and lymphoid PB chimerism 20 weeks after transplantation. Results are expressed as mean ± SD (n = 7-8 from 2 independent experiments). Two-way ANOVA with Sidak’s post hoc test; *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.

Impact of constitutive AKT activation on HSC function in obesity. (A) Gene set enrichment analysis (GSEA) for upregulated genes in db HSC-SLAMs. Numbers in parentheses show the number of genes in indicated gene sets. (B) Scheme of manually curated upregulated (blue) and downregulated (green) genes in HSC-SLAMs identified by genome-wide gene expression analysis and linked to the phosphatidylinositol pathway and AKT signaling. (C) Representative fluorescence-activated cell sorting (FACS) plot and histogram (left panel) showing the gating strategy and levels of AKT and STAT3 phosphorylation in the HSC-SLAM compartment isolated from the BM of 4-month-old control and db mice. Right panel shows relative average mean fluorescence intensity (MFI) ± standard deviation (SD) of AKT, STAT3, STAT5, and pERK phosphorylation in db HSCs compared with control (n = 5). Student t test; *P ≤ .05. (D) Experimental scheme for in vivo pharmacologic AKT inhibition (left panel). Right panel shows relative average MFI ± SD of AKT phosphorylation in each experimental group (n = 3). Two-way ANOVA with Tukey’s post hoc test; *P ≤ .05; **P ≤ .01. (E) Mean percentage ± SD of HSC-SLAMs and MPPs (LSK CD48+) in the BM of 4-month-old control and db mice treated with vehicle (veh) or the AKT inhibitor MK-2206 for 2 weeks (n = 12-20 from 5 independent cohorts). Two-way ANOVA with Tukey’s post hoc test; ****P ≤ .0001. (F) Representative FACS histogram (left panel) and average MFI ± SD (right panel) showing levels of CD34 marker on HSC-SLAMs isolated from 4-month-old control and db mice treated with vehicle or the AKT inhibitor MK-2206 for 2 weeks (n = 11-12 from 5 independent cohorts). Two-way ANOVA with Tukey’s post hoc test; ***P ≤ .0005. (G) PB chimerism in competitive reconstitution assays using purified HSC-SLAMs isolated from the BM of control and db mice treated with vehicle or the AKT inhibitor MK-2206 for 2 weeks. The right graph shows myeloid and lymphoid PB chimerism 20 weeks after transplantation. Results are expressed as mean ± SD (n = 7-8 from 2 independent experiments). Two-way ANOVA with Sidak’s post hoc test; *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.

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