Fig. 6: FoxO1 signaling downstream of TLR4 induces CHFR expression. | Nature Communications

Fig. 6: FoxO1 signaling downstream of TLR4 induces CHFR expression.

From: Ubiquitin ligase CHFR mediated degradation of VE-cadherin through ubiquitylation disrupts endothelial adherens junctions

Fig. 6

a Schematic showing putative transcription factor binding sites in the promoter regions of mChfr, hCHFR, mFoxO1, and hFoxO1. b, c WT mice were challenged with LPS (i.p; 10 mg/kg body weight) for different times. b Lungs harvested were used for total RNA isolation followed by RT-qPCR. Shown are mean values ± SEM (n = 3 mice/group, one-way ANOVA followed by Tukey’s post-hoc test). c Lungs were used for IB to determine expression of FoxO1. Shown are mean values ± SEM (n = 3 mice/group; one-way ANOVA followed by Tukey’s post-hoc test). d WT mice lung ECs challenged with LPS (5 μg/ml) for different time intervals were used for ChIP assay to determine FoxO1 binding to mChfr promoter. Shown are mean values ± SEM. FoxO1-BS, FoxO1 binding sites; (n = 3 independent experiments; one-way ANOVA followed by Tukey’s post-hoc test). e, f WT mice challenged with LPS as in (b) and then lungs harvested were used for total RNA isolation followed by RT-qPCR and IB to determine CHFR expression. Shown are mean values ± SEM (e–f, n = 3 mice/group; one-way ANOVA followed by Tukey’s post-hoc test). g WT mice were injected with DMSO or the FoxO1 inhibitor (AS1842856; 5 mg/kg, i.p. one injection per day for 4 days) and then challenged with LPS (i.p.;10 mg/kg body weight) or saline for 6 h. Lungs harvested were used for IB analysis to determine expression of Chfr and ICAM-1. Results show a representative blot. Shown are mean values ± SEM (n = 3 mice/group; two-way ANOVA followed by Tukey’s post-hoc test). h, i Augmented expression of CHFR and FoxO1 in lung endothelial cells of patients with acute respiratory distress syndrome (ARDS). Lung sections from non-ARDS control and sepsis/ARDS patients were stained with EC marker vWF (red) and the E3 ligase CHFR (green) (h) or FoxO1 (green) (i). DAPI (blue). Shown are mean values ± SEM (n = 5 samples from each group; unpaired two-tailed Student’s t test). Fluorescent intensity is presented as arbitrary units (arb. units). V, blood vessel.

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