Fig 2: FoxP4 directly controls UCP1 gene expression. A: FoxP4 and UCP1 mRNA and protein levels in 10T1/2 differentiated cells with either FoxP4 overexpression or knockdown. B: In silico analysis using the Genomatix program identified a putative Forkhead-binding motif at -479 to -472 from the UCP1 transcription start site. C: ChIP assays performed in 10T1/2 differentiated cells with either FoxP4 overexpression or knockdown. Anti-IgG and anti-FoxP4 antibodies were used to assess the occupancy of FoxP4 at the putative Forkhead response element identified in the UCP1 promoter. D: Luciferase assay in HEK-293 and differentiated 10T1/2 cells cotransfected with either vector or FoxP4, in the presence of the UCP1 luciferase reporter construct. Results show a representative experiment performed in triplicate. Results are expressed as a mean ± SEM from three independent experiments. *P < 0.05; **P < 0.005; and ***P < 0.001. FoxP4, Forkhead box P4; HEK-293, human embryonic kidney 293 cells; UCP1, uncoupling protein 1.

Fig 3: FoxP4 levels are induced by thermogenic stimuli. A–C: Relative FoxP4 mRNA levels in differentiated 10T/2 cells treated for 4 h with different concentrations of forskolin (A), isoproterenol (B), and IBMX indicated in the graph. C: Relative FoxP4 mRNA (D) and protein levels (E) in differentiated scWAT SVF cells treated with 10 µM forskolin. F–I: Relative FoxP4 mRNA levels in BAT and scWAT of 9-week-old male and female mice (n = 4) treated with CL316, 243, or vehicle, for 3 h (F and G) or exposed to cold or room temperature for 6 h (H and I). Results are expressed as mean ± SEM from three independent experiments. *P < 0.05; **P < 0.005; and ***P < 0.001. BAT, brown adipose tissue; FoxP4, Forkhead box P4; IBMX, isobutylmethylxanthine; scWAT, subcutaneous white adipose tissue; SVF, stromal vascular fraction.

Fig 4: Identification of target genes of miR-423-5p. (A-C) The location of miR-423-5p and FOXP4 mRNA in BEAS-2B cells was determined by fluorescence in situ hybridization (scale bar=200 µm). (D) The binding of miR-423-5p and FOXP4 in BEAS-2B cells was detected by an RNA pull-down assay. (E) The expression of FOXP4 in AF tissues and TGF-ß1-stimulated BEAS-2B cells was detected by immunohistochemistry (scale bar=50 µm) and RT-qPCR. The relative expression of FOXP4 in BEAS-2B cells transfected with a miR-423-5p mimic or inhibitor for 24 h and then treated with TGF-ß1 for 24 h was determined by (G) RT-qPCR, (F) immunofluorescence staining (scale bar=50 µm.) and (H) western blot analysis. *P<0.05 and **P<0.01. miR, microRNA; FOXP4, forkhead box p4; RT-qPCR, reverse transcription-quantitative PCR; AF, airway fibrosis.

Fig 5: miR-423-5p promotes TGF-ß1-induced EMT through the PI3K/AKT/mTOR signaling pathway. (A) The protein expression of PI3K, AKT and mTOR was detected by immunohistochemistry (scale bar=50 µm.). (B) The relative expression of p-PI3K, p-AKT and p-mTOR in BEAS-2B cells transfected with a miR-423-5p mimic or inhibitor for 24 h and then treated with TGF-ß1 for 24 h was determined by western blot analysis. (C) The relative expression of p-PI3K, p-AKT and p-mTOR was determined by western blot analysis. BEAS-2B cells transfected with siRNA-FOXP4 to silence FOXP4 were transfected with a miR-423-5p inhibitor for 24 h and then treated with TGF-ß1 for 24 h. *P<0.05 and **P<0.01. miR, microRNA; EMT, epithelial-mesenchymal transition; p-, phosphorylated; si, short interfering.