Journal: Nucleic Acids Research

Article Title: FOXN3 integrates the KU70/KU80/SREBP-1 complex to regulate lipid metabolism in non-alcoholic fatty liver disease

doi: 10.1093/nar/gkag171

Figure Lengend Snippet: FOXN3 and the KU70/KU80/SREBP-1 complex colocalize at the promoters of SREBP-1 response genes. ( A ) Mass spectrometry analysis of HEK293T cells transfected with Flag-tagged FOXN3 reveals the number of unique peptides associated with FOXN3. The representative peptide sequences of KU70 and KU80, which were immunoprecipitated by Flag-tagged FOXN3, are presented. ( B ) An anti-Flag Co-IP assay was performed in HEK293T cells transfected with the specified plasmids to investigate the association of Flag-tagged FOXN3 with the KU70/KU80/SREBP-1 complex (P: precursor of SREBP-1). ( C ) Anti-FOXN3 Co-IP analysis was performed in HepG2 cells to examine the endogenous association between FOXN3 and KU70/KU80/SREBP-1 complex. (P: precursor of SREBP-1; N: nuclear form of SREBP-1). ( D ) Anti-Flag Co-IP assays were performed in HepG2 cells to detect the association of Flag-tagged FOXN3 with the KU70/KU80/SREBP-1 complex following treatment with FFA (400 μM) at the indicated time points. The cells were treated with MG-132 (20 μM, 4 h) prior to collection (P: precursor of SREBP-1; N: nuclear form of SREBP-1). ( E ) Venn diagrams showing the overlapping peaks identified by anti-KU80, anti-FOXN3, and anti-SREBP-1 CUT&Tag analyses in HepG2 cells treated with FFA (400 μM, 24 h). ( F ) Venn diagrams displaying the number of genes co-targeted by KU80, FOXN3, and SREBP-1 based on CUT&Tag analyses in HepG2 cells treated with FFA (400 μM, 24 h). ( G ) Density distributions (normalized read densities) of the mapped reads from anti-KU80, anti-FOXN3, and anti-SREBP-1 CUT&Tag analyses at the transcription start site (TSS) are shown. ( H ) Genomic distribution of the transcriptional targets of KU80, FOXN3, and SREBP-1 in HepG2 cells treated with FFA (400 μM, 24 h), as determined via CUT&Tag data analysis. ( I ) The CUT&Tag data analysis in HepG2 cells treated with FFA (400 μM, 24 h) displays the binding profiles of KU80, FOXN3, and SREBP-1 to representative SREBP-1 response genes. The graphs represent the proportion of reads enriched in the peak region relative to every one million total reads. The blotting data D was quantified as the mean fold change from two independent experiments using ImageJ software and was analyzed using two-tailed Student’s t -tests; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies utilized were as follows: anti-SREBP-1 (14088-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-His (66005-1-Ig, Proteintech), anti-KU70 (10000-0-AP, Proteintech), anti-FOXN3 (25399-1-AP, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-KU70 (10723-1-AP, Proteintech), anti-KU80 (16389-1-AP, Proteintech), anti-β-actin (20536-1-AP, Cell Signaling Technology), anti-FASN (10624-2-AP, Proteintech), anti-SCD1 (28678-1-AP, Proteintech), anti-LDLR (10785-1-AP, Proteintech), anti-HMGCS1 (17643-1-AP, Proteintech), anti-SREBP-1 (66875-1-Ig, Proteintech, for IP), anti-rabbit IgG (30000-0-AP, Proteintech), and anti-FOXN3 (ab129453, Abcam).

Techniques: Mass Spectrometry, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Software, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: FOXN3 integrates the KU70/KU80/SREBP-1 complex to regulate lipid metabolism in non-alcoholic fatty liver disease

doi: 10.1093/nar/gkag171

Figure Lengend Snippet: Depletion of FOXN3 or KU70/KU80 suppresses SREBP-1 transcriptional activity by disrupting the recruitment of SREBP-1 to its target gene elements. ( A ) Anti-SREBP-1 CUT&Tag analysis in HepG2 cells treated with FFA (400 μM, 24 h) was performed to examine the impact of FOXN3 knockdown on the binding profiles of SREBP-1 to lipogenic response genes. The graphs represent the proportion of reads enriched in the peak region relative to every one million total reads. ( B ) ChIP analysis was conducted in HepG2 cells treated with FFA (400 μM, 24 h) to assess the effect of FOXN3 knockout on the distribution of SREBP-1 at lipogenic response gene promoters. ( C ) ChIP analysis was conducted in HepG2 cells treated with FFA (400 μM, 24 h) to assess the effect of KU70 and KU80 knockdown on the distribution of SREBP-1 at lipogenic response gene promoters. ( D ) ChIP analysis was conducted in HepG2 cells treated with FFA (400 μM, 24 h) to assess the effect of KU70 and KU80 knockdown on the distribution of FOXN3 at lipogenic response gene promoters. ( E ) qPCR analysis was conducted in HepG2 cells treated with FFA (400 μM, 24 h) to assess the effect of KU70 and KU80 knockdown on the levels of lipogenic response genes. ( F ) A anti-Flag Co-IP assay was performed in FFA-treated (400 μM, 24 h) HepG2 cells stably expressing Flag-tagged FOXN3 to assess the effect of KU70 or KU80 knockdown on the interaction between FOXN3 and nuclear SREBP-1. The cells were treated with MG-132 (20 μM, 4 h) prior to collection. ( G ) Anti-KU70 Co-IP analysis was conducted in WT and FOXN3-knockout HepG2 cells treated with FFA (400 μM, 24 h) to assess the impact of FOXN3 deficiency on the association between KU70 and nuclear SREBP-1. ( H ) A anti-Flag Co-IP assay was performed in FFA-treated (400 μM, 24 h) HepG2 cells transfected with Flag-tagged KU80 to assess the effect of FOXN3 knockout on the interaction between KU80 and nuclear SREBP-1. ( I ) A anti-Flag Co-IP assay was performed in HepG2 cells transfected with the indicated plasmids to assess the effect of His-tagged KU70 overexpression on the interaction between FOXN3 and nuclear SREBP-1. The cells were treated with MG-132 (20 μM, 4 h) prior to collection. ( J ) A anti-Flag Co-IP assay was performed in HepG2 cells transfected with the indicated plasmids to assess the effect of HA-tagged FOXN3 overexpression on the interaction between KU80 and nuclear SREBP-1. The data B–E were assessed via two-tailed Student’s t -tests and are shown as means ± S.D. The blotting data F–J were quantified as the mean fold change and subjected to two-tailed Student’s t -tests; * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The primary antibodies utilized were as follows: anti-SREBP-1 (14088-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-His (66005-1-Ig, Proteintech), anti-KU70 (10000-0-AP, Proteintech), anti-FOXN3 (25399-1-AP, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-KU70 (10723-1-AP, Proteintech), anti-KU80 (16389-1-AP, Proteintech), anti-β-actin (20536-1-AP, Cell Signaling Technology), anti-FASN (10624-2-AP, Proteintech), anti-SCD1 (28678-1-AP, Proteintech), anti-LDLR (10785-1-AP, Proteintech), anti-HMGCS1 (17643-1-AP, Proteintech), anti-SREBP-1 (66875-1-Ig, Proteintech, for IP), anti-rabbit IgG (30000-0-AP, Proteintech), and anti-FOXN3 (ab129453, Abcam).

Techniques: Activity Assay, Knockdown, Binding Assay, Knock-Out, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Transfection, Over Expression, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: FOXN3 integrates the KU70/KU80/SREBP-1 complex to regulate lipid metabolism in non-alcoholic fatty liver disease

doi: 10.1093/nar/gkag171

Figure Lengend Snippet: Inhibition of FOXN3 phosphorylation at S83 and S85 enhances SREBP-1 transcriptional activity. ( A ) Western blot analysis was performed to assess the levels of total FOXN3 and its phosphorylation at S83 and S85 in the livers of mice administered a HFD for 0, 12, and 20 weeks. ( B–D ) Western blot analysis was performed to assess the levels of total FOXN3 and its phosphorylation at S83 and S85 in HepG2 (B) and Huh7 (C) cells, as well as in MPHs (D) exposed to FFA treatment at the indicated time points. ( E ) A luciferase assay was conducted in FOXN3-knockout HepG2 cells to assess the effect of WT FOXN3 and its S83,85A mutant on the transcriptional activity of SREBP-1. A plasmid containing an SREBP element was co-transfected with a plasmid expressing either Flag-tagged WT FOXN3 or its S83,85A mutant. The cells were treated with FFA (400 μM, 24 h) prior to collection. ( F ) qPCR analysis was conducted to measure the RNA levels of lipogenic response genes in FOXN3-knockout HepG2 cells infected with lentivirus expressing either Flag-tagged WT FOXN3 or its S83,85A mutant. The cells were treated with FFA (400 μM, 24 h) prior to collection. ( G ) Western blot analysis was conducted to measure the protein levels of lipogenic response genes in FOXN3-knockout HepG2 cells infected with lentivirus expressing either Flag-tagged WT FOXN3 or its S83,85A mutant. The cells were treated with FFA (400 μM, 24 h) prior to collection. ( H ) Anti-SREBP-1 and anti-Flag ChIP analyses were conducted to measure the binding capacity of SREBP-1 to the promoters of lipogenic response genes in FOXN3-knockout HepG2 cells infected with lentivirus expressing either Flag-tagged WT FOXN3 or its S83,85A mutant. The cells were treated with FFA (400 μM, 24 h) prior to collection. ( I ) A Co-IP assay was performed in FOXN3-knockout HepG2 cells transfected with Flag-tagged WT FOXN3 or its S83,85A mutant to assess their interaction with the KU70/KU80/SREBP-1 complex. The cells were treated with MG-132 (20 μM, 4 h) prior to collection. The data in E, F, and H were assessed via two-tailed Student’s t -tests and are shown as means ± S.D. The blotting data A–D, J, and I were quantified as the mean fold change and subjected to two-tailed Student’s t -tests; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies utilized were as follows: anti-SREBP-1 (14088-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-His (66005-1-Ig, Proteintech), anti-KU70 (10000-0-AP, Proteintech), anti-FOXN3 (25399-1-AP, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-KU70 (10723-1-AP, Proteintech), anti-KU80 (16389-1-AP, Proteintech), anti-β-actin (20536-1-AP, Cell Signaling Technology), anti-FASN (10624-2-AP, Proteintech), anti-SCD1 (28678-1-AP, Proteintech), anti-LDLR (10785-1-AP, Proteintech), anti-HMGCS1 (17643-1-AP, Proteintech), anti-SREBP-1 (66875-1-Ig, Proteintech, for IP), anti-rabbit IgG (30000-0-AP, Proteintech), and anti-FOXN3 (ab129453, Abcam).

Techniques: Inhibition, Phospho-proteomics, Activity Assay, Western Blot, Luciferase, Knock-Out, Mutagenesis, Plasmid Preparation, Transfection, Expressing, Infection, Binding Assay, Co-Immunoprecipitation Assay, Two Tailed Test