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