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. Author manuscript; available in PMC: 2021 Sep 15.
Published in final edited form as: Nat Metab. 2021 Mar 15;3(3):318–326. doi: 10.1038/s42255-021-00356-0

FIGURE 3. Fasting-induced FOXO4 Effects.

FIGURE 3.

a, FOXO4 transcription network and its targets identified by GeneMANIA analysis. The 24-hr. fasting DE genes (circular nodes) show fold increased (blue circles) or decreased (red circles) expression relative to refeeding levels. Validation of target relative genes transcript levels by qRT-PCR in PBMCs (bar plots mean ± SEM with mean values of 3–4 replicates from n=14 subjects, two-sided, paired Student’s t-test). b, Concurrent analysis using the Find Individual Motif Occurrences (FIMO) tool identified the FOXO4 binding motif in the promoter regions of a significant proportion of fasted DE upregulated genes (fisher exact test for FOXO4 motif enrichment significance, p=0.049). c, Representative protein blots from 3 subjects (left panel) and quantitative changes (right panel) of FOXO4 levels in CD4+ T cells from the 3 caloric-load states. (Bar graph mean ± SEM, n=7 subjects, two-sided, paired Student’s t-test). d-e, Cytokine release of IFNγ, IL-4 and IL-17 following FOXO4 knockdown (KD) and overexpression in activated CD4+ T cells. The cytokine release of KD cells measured (n=5 separate experiment (d)) or from FOXO4-overexpressed cells (n=10 separate experiments (e)) using two-sided, paired Student’s t-test analysis. The dot and line plots show the mean value of each subject and the values represent the average of duplicate experiments. Statistical analysis using two-sided, paired Wilcoxon test. f-g, Representative protein blot (left panel) and quantification (right panel) comparing transduction of control vs. FOXO4 lentivirus (OE) with (+) or without (−) CD4+ T cell activation (two-sided, paired Student’s t-test). f, mTOR signaling pathway phosphorylation and total protein kinase levels (Bar graphs - mean ± SEM of n=6 biologically independent subjects). g, STAT signaling proteins (Bar graphs - mean ± SEM of 6–8 biologically independent subjects). h-I, Flow cytometric plots of cell surface; Th1 (CXCR3+CCR6−), Th17 (CXCR3−CCR6+), and Th2 (CD294+) and intracellular; IFNγ, IL-17, and IL-4, markers from healthy volunteer CD4+ T cells transduced with control or FOXO4 lentivirus (Bar graphs - mean ± SEM from n=8 subjects, two-sided, paired Wilcoxon test). Supplementary Data2 for Fig.3 shows flow cytometry gating strategy.

Source Data Fig.3 Unprocessed immunoblots.