Direct delivery of designed protein inhibitors of b ZIP transcription factor for studying preadipocyte differentiation in NIH 3T3 L1 cell lines

Abstract

Adipogenesis is a time-dependent process involving number of transcription factors, regulators and enhancers. The expression of each factor is crucial for series of events during differentiation process. Inhibition of initial factors may hinder this time-dependent process. In this study, we delivered purified dominant-negative A-C/EBP protein, designed to inhibit the DNA binding of C/EBP protein, to differentiating 3T3-L1 cells at different time points and observed the outcomes. Protein delivery is compared with A-C/EBP encoding plasmid delivery, as later took approximately 48 hours for translated product to appear and inhibit its targets. The inhibition of C/EBPand#946; was observed within hours of A-C/EBP protein transfection as compared to gene delivery. Liposome-mediated transfection was successful in delivering anionic A-C/EBP tagged with EGFP into the cell cytoplasm. Real-time PCR and immunoblotting of different factors involved in adipogenesis have shown the downregulation of C/EBPand#946; related genes and proteins. ChIP results showed the lesser binding of C/EBPand#946; to the promoter-region of adipocyte-marker genes in transfected cells. Gene expression of preadipocyte-marker genes in protein transfected cells was higher as compared gene transfected cells which is a new finding. Our study shows similar results as found with gene delivery but in lesser time span and protein was degraded with time so there will be no long-term off-target effects. Also, the higher expression of preadipocyte-markers in protein transfected cells proves the non-conversion of preadipocytes to adipocytes. Thereby, direct delivery of A-C/EBP protein in differentiating 3T3-L1 cells can change the fate of ongoing process of adipogenesis. newline