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 Stat5a and Creb were downregulated in A-C/EBP protein samples. Five preadipocyte markers, namely, Pdgfrα, Pdgfrβ, Ly6A, CD34, and 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/EBPβ protein to understand time-dependent preadipocytes differentiation.
Transformation of committed 3T3-L1 preadipocytes to lipid-laden adipocytes involves timely appearance of numerous transcription factors (TFs), foremost among them is 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 tagged A-C/EBP were observed in 3T3-L1 cells within 2hrs of protein inhibitor transfections whereas for gene transfection signals appeared in 48hrs. Following transient transfections, expression profiles of 24 marker genes belonging to pro-and anti-adipogenic, cell cycle, and preadipocytes pathways was analyzed. 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 protein and 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 protein samples whereas only Ly6A and CD34 were expressed in gene transfected samples. Pdgfrα and Pdgfrβ, two known cell fate markers were expressed in protein transfected samples 5-days post-differentiation suggesting a possible reversal of differentiation. Our study provides evidences for the robust and efficient knockdown of C/EBPβ protein to understand time-dependent gene regulation during adipogenesis.
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