Figure 1.

The TanFe (transcellular activator of nuclear FOXF1 expression) small-molecule compound increases FOXF1 (Forkhead Box F1) protein amounts in vitro. (A) Chemical structure of the TanFe compound. (B) Western blots show amounts of endogenous FOXF1 protein and other transcription factors in TanFe-treated fetal lung endothelial Mouse Fetal Lung Mesenchyme-91 U (MFLM-91U) cells. Cells were treated with either vehicle alone (control) or 20 μM of TanFe and harvested 24 hours after the treatment. Protein samples were pooled from five independent cell cultures (left panels). Three independent Western blots were quantified and normalized to β-Actin (right graph). (C) TanFe increases nuclear FOXF1 fluorescence. MFLM-91 U endothelial cells were fixed and stained for FOXF1 (green). DAPI was used to visualize cell nuclei (blue). Inserts show high-magnification images of FOXF1-stained cells. (D and E) Western blots show the dose response and the time course of TanFe treatment in MFLM-91 U cells (n = 3 independent experiments). Half maximal effective concentration (EC₅₀) for TanFe is 3.6 μM (dotted lines). (F) Luciferase (Luc) assay shows increased FOXF1 transcriptional activity in TanFe-treated MFLM-91 U cells. Cells were transfected with either FOXF1-specific LUC reporter plasmid (FOXF1-LUC) or Empty-LUC plasmid (control) and treated with TanFe for 24 hours (n = 5 for each group). (G) TanFe treatment does not change Foxf1 mRNA. Quantitative RT-PCR was used to measure Foxf1 mRNA in MFLM-91 U cells after treatment with different concentrations of TanFe (n = 3). Expression levels were normalized to β-Actin (Actb mRNA). *P < 0.05 and ****P < 0.0001. FACT 140 = facilitates chromatin transcription complex subunit SPT16 -140 kDa; NF-κB = nuclear factor-κB; n.s. = not significant.