Transformation of committed 3T3-L1 preadipocytes to lipid-laden adipocytes involves the timely appearance of numerous transcription factors (TFs); foremost among them, C/EBPβ is expressed during the 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 2 h of transfections, whereas for A-C/EBP gene transfections, equivalent signals appeared in 48 h. Following transient transfections, the expression profiles of 24 marker genes belonging to pro- and anti-adipogenic, cell cycle, and preadipocyte pathways were analyzed. Expectedly, the 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 and were downregulated in A-C/EBP protein samples. Five preadipocyte markers, namely, , , , and , showed high expression in A-C/EBP protein samples, whereas only and were expressed in A-C/EBP gene-transfected samples. and , 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/EBPβ protein to understand time-dependent preadipocytes differentiation.
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| http://dx.doi.org/10.3389/fmolb.2020.603168 | DOI Listing |
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