Fig. 4: FOXN3 facilitates β-TrCP-mediated Smad4 degradation through enhancing their interaction.

a An Anti-Flag Co-IP assay was conducted in HEK293T cells transfected with Flag-tagged Smad4 to investigate the association of Smad4 with endogenous β-TrCP, hnRNPU, and FOXN3. The cells were collected 48 h post-transfection. TCL: total cell lysate. b An anti-Smad4 Co-IP assay was performed in A549 cells to detect the Smad4 association with endogenous β-TrCP, hnRNPU, and FOXN3. c An anti-Flag Co-IP assay was conducted to detect the interaction between Flag-tagged FOXN3 and both Smad4 and hnRNPU in A549 cells following TGF-β treatment (10 ng/ml, 16 hr) at the indicated time points. The cells infected with lentivirus expressing Flag-tagged FOXN3 were treated with 0.05% FBS for 8 h prior to TGF-β treatment and then collected 48 h post-infection. d The effect of FOXN3 overexpression on Flag-tagged Smad4 ubiquitination in HEK293T cells was detected by a ubiquitination assay. The cells transfected with indicated combinations of HA-tagged FOXN3, Flag-tagged Smad4, Myc-tagged ubiquitin, and shRNA targeting β-TrCP were treated with MG-132 (20 μM, 4 h) and collected 48 h post-transfection. e Western blot analysis was carried out in A549 cells to examine the impact of FOXN3 overexpression on the stability of Smad4 in the presence or absence of β-TrCP. Lentiviral infection was used to transduce HA-tagged FOXN3 and β-TrCP shRNA into the cells, which were collected 48 h post-infection. f Western blot analysis was carried out in primary pulmonary fibroblasts to examine the impact of FOXN3 overexpression on the stability of Smad4 in the presence or absence of β-TrCP. Lentiviral infection was utilized to transduce HA-tagged FOXN3 and β-TrCP shRNA into the cells, which were collected 48 hrs post-infection. g A Co-IP assay was conducted to assess the impact of lentivirus-mediated FOXN3 overexpression on the association of endogenous Smad4 with hnRNPU and β-TrCP in A549 cells. Before collection, the cells infected with lentivirus expressing Flag-tagged FOXN3 were treated with MG-132 (20 μM, 4 h) and collected 48 hrs post-infection. h An anti-Flag Co-IP assay was performed to detect the effect of FOXN3 forkhead domain deficiency on the interaction between Flag-tagged Smad4 and both hnRNPU and β-TrCP in HEK293T cells. Prior to collection, the cells transfected with indicated combinations of HA-tagged WT FOXN3 or its forkhead-deficient mutant, Flag-tagged Smad4, and Myc-tagged β-TrCP (ΔF) were treated with MG-132 at a concentration of 20 μM for 2 h and collected 48 hrs post-transfection. i Quantitative PCR analysis showing the RNA levels of pro-fibrotic factors in A549 cells overexpressing FOXN3 WT or its forkhead domain-deficient mutant through lentiviral infection. The cells were collected 48 hrs post-infection. The data (i) was assessed by one-way ANOVA and was shown as the mean ± SD. The blotting data (a–h) are representative of two independent experiments and were quantified using ImageJ software.