Fig. 3: FOXN3 promotes the instability of Smad4 and blocks Smad-mediated transcriptional response.

a A volcano plot illustrating the differential expression of genes in the lungs of mice following FOXN3 overexpression. b Gene ontology analysis was performed on the RNA sequencing data obtained from the lungs of both WT and Fxon3^LSL KI mice. GO enrichment analysis of the differentially expressed genes was conducted using the Wallenius non-central hypergeometric distribution, which accounts for gene length bias. A p-value threshold of ≤ 0.05 was established for enrichment significance. c KEGG and heatmap analyses of the RNA sequencing data were conducted to illustrate the expression changes of Smad downstream-regulated genes. d Immunoblot analysis was conducted to assess the effect of siRNA-mediated FOXN3 knockdown on the levels of Smad proteins in both A549 and primary ATII cells upon TGF-β treatment (10 ng/ml, 16 hr). The cells were treated with 0.05% fetal bovine serum (FBS) for 8 h prior to TGF-β treatment and collected 48 hrs post-siRNA transfection. TCL: total cell lysate. e Immunoblot analysis was conducted to assess the protein expression levels of Smads and FOXN3 in the lungs of both WT and Fxon3^LSL KI mice (n = 4). (f–h) ChIP assays were conducted in A549 cells with or without TGF-β treatment (10 ng/ml, 16 hr) to investigate the effect of siRNA-mediated FOXN3 knockdown on the transcriptional recruitment of Smad proteins to the promoters of their target genes. The cells were collected 48 hrs post-transfection. Representative data from three independent experiments (n = 3). i ChIP assays were conducted in A549 cells with or without TGF-β treatment (10 ng/ml, 16 hr) to examine the distribution of FOXN3 on the promoters of Smad target genes. Representative data from three independent experiments (n = 3). The data (f–h) were assessed by one-way ANOVA, and the data (i) was assessed by a two-tailed Student’s t test. All data was shown as the mean ± SD. The blotting data (d) are representative of two independent experiments and were quantified using ImageJ software.