Fig. 8: The phosphorylation of FOXN3 by NEK6 promotes its degradation, thereby enhancing the association of the Smad complex with chromatin for transcriptional activation.

a A Co-IP assay was conducted in HEK293T cells to detect the impact of NEK6 overexpression on the interaction between Flag-tagged WT FOXN3 or its S412,416 A mutant and Myc-tagged β-TrCP. The cells transfected with the indicated combinations were collected 48 hrs post-transfection. b Bacterially expressed NEK6 and either WT FOXN3 or its S412,416 A mutant were subjected to an in vitro kinase assay, followed by immunoblot analysis of FOXN3 S412 and S416 phosphorylation with a phosphospecific antibody. c A ubiquitination assay was performed in HEK293T cells to detect the impact of NEK6 overexpression on the ubiquitination of WT FOXN3 or its S412,416 A mutant. The cells transfected with the indicated combinations of Flag-tagged FOXN3, HA-tagged NEK6, Myc-tagged β-TrCP, and HA-tagged ubiqutin were treated with MG-132 (20 μM, 4 h) prior to collection 48 hrs post-transfection. d A ubiquitination assay was performed in HEK293T cells to detect the impact of lentivirus-mediated NEK6 overexpression on the ubiquitination of endogenous Smad4. The cells transfected with Flag-tagged WT FOXN3 or its S412,416 A mutant and HA-tagged NEK6 were treated with MG-132 (20 μM, 4 hr) prior to collection 48 hrs post-transfection. e Immunoblot analysis in HEK293T cells showing the effect of NEK6 overexpression on the levels of FOXN3 and Smad4. The cells transfected with the indicated combinations of Flag-tagged Smad4, HA-tagged FOXN3, HA-tagged NEK6, and Myc-tagged β-TrCP were collected 48 hrs post-transfection. f A Co-IP assay was conducted in HEK293T cells to detect the effect of NEK6 overexpression on the interaction between Flag-tagged Smad4 and Myc-tagged β-TrCP in the presence of WT FOXN3 or its S412,416 A mutant. The cells were treated with MG-132 (20 μM, 2 hr) prior to collection 48 hrs post-transfection. g Immunoblot analysis was performed to assess the impact of NEK6 knockdown on the levels of indicated proteins in primary ATII cells in response to TGF-β treatment (10 ng/ml, 16 hr). The cells were treated with 0.05% FBS for 8 h prior to TGF-β treatment and then collected 48 h post-siRNA transfection. h Immunoblot analysis was conducted in primary pulmonary fibroblasts isolated from WT or Foxn3 KI mice to demonstrate the effects of NEK6 knockdown on the levels of FOXN3, its phosphorylation, and Smad4. All cells were treated with 0.05% FBS for 8 h, followed by TGF-β treatment (10 ng/ml, 16 hr), and then collected 48 h post-siRNA transfection. i, j Step-wise fractionation analysis was performed in both A549 (i) and primary ATII (j) cells to assess the effect of NEK6 knockdown on the association of Smad complex with chromatin. All cells were treated with 0.05% FBS for 8 h, followed by TGF-β treatment (10 ng/ml, 16 hr), and then collected 48 h post-siRNA transfection. k, l Quantitative PCR analysis was conducted in both A549 (k) and primary ATII (l) cells to assess the effect of NEK6 knockdown on Smad transcriptional activity. All cells were treated with 0.05% FBS for 8 h, followed by TGF-β treatment (10 ng/ml, 16 hr), and then collected 48 h post-siRNA transfection. m A proposed working model elucidates the transcriptional activation of Smad signaling through NEK6-mediated FOXN3 degradation. The schematic representation was created in BioRender. LIAN, J. (2024) https://BioRender.com/w42l861. The data (k and l) were assessed by a two-tailed Student’s t test. All data are shown as the mean ± SD. The blotting data (a–j) are representative of two independent experiments and were quantified using ImageJ software.