AUTHOR=Sharma Nishtha , Kaur Raminder , Yadav Binduma , Shah Koushik , Pandey Harshita , Choudhary Diksha , Jain Prateek , Aggarwal Aanchal , Vinson Charles , Rishi Vikas 

TITLE=Transient Delivery of A-C/EBP Protein Perturbs Differentiation of 3T3-L1 Cells and Induces Preadipocyte Marker Genes

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=Volume 7 - 2020

YEAR=2021

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2020.603168

DOI=10.3389/fmolb.2020.603168

ISSN=2296-889X

ABSTRACT=Transformation of committed 3T3-L1 preadipocytes to lipid-laden adipocytes involves timely appearance of numerous transcription factors (TFs), foremost among them C/EBPβ, is expressed during early phases of differentiation. Here we describe liposome-mediated protein transfection approach to rapidly downregulate C/EBPβbyA-C/EBP protein inhibitor.SignalsfromEGFP-tagged A-C/EBP protein were observed in 3T3-L1 cells within 2hrs of transfections whereas for A-C/EBP gene transfections equivalent signals appeared in 48hrs. Following transient  transfections, expression profiles of 24 marker genes belonging to pro-and anti-adipogenic, cell cycle, and preadipocytes pathways were analyzed. Expectedly, mRNA and protein expression profiles of  adipocyte-marker genes showed lower expression in both A-C/EBP protein and gene transfected samples. Interestingly, for preadipocytes and cell fate determinant genes, striking differences were observed between  A-C/EBP protein and A-C/EBP gene transfected samples. Preadipocyte differentiation factors Stat5a and Creb  were downregulated in A-C/EBP protein samples. Five preadipocyte markers, namely, Pdgfrα, Pdgfrβ, Ly6A, CD34, Itgb1 showed high expression in A-C/EBP protein samples whereas only Ly6A and CD34 were expressed in A-C/EBP gene transfected samples.  Pdgfrα and Pdgfrβ, two known cell fate markers were expressed in A-C/EBP protein transfected samples suggesting a possible reversal of differentiation. Our study provides evidences for the immediate and efficient knockdown of C/EBP protein to understand time-dependent preadipocytes differentiation.