Fig. 6

Zebrafish Nkx1-2 binds to cebpa promoter and activates its transcription. (A) The effect of zebrafish nkx1.2 overexpression on cebpa expression during 3T3-L1 differentiation. The beta-actin gene was used as loading control. (B) Effect of nkx1.2 knockout on cebpa expression during 3T3-L1 differentiation. The beta-actin gene was used as loading control. (C) Cebpa promoter activity analysis. The reporter gene vector (pGL3 promoter) and the internal reference plasmid (pRL-TK) containing promoter sequences of different lengths were co-transfected into HEK293T cells. After 48 h, a double luciferase reporter gene detection experiment was performed. C2000, C1000, and C500 represent 2000 bp, 1000 bp, and 500 bp of pGL3-cebpa-promoter, respectively. (a) 2000 bp, 1000 bp, and 500 bp diagrams of C/EBPα promoter. (b) C/EBPα promoter activity detection. (D) Regulating effect of full-length and truncated Nkx1-2 on cebpa promoter by the double luciferase reporter gene assay. (E) Regulation analysis of truncated cebpa promoter by Nkx1-2. HEK 293T cells were co-transfected with pcDNA3.1-nkx1.2 expression vector and pGL3-cebpa-promoter reporter gene vector, and the activity of dual luciferase was detected 48 h later. (a) 500 bp, 1000 bp, 1500 bp, 1800 bp, and 2000 bp diagrams of cebpa promoter. (b-f) Activity detection of different lengths of cebpa promoter by Nkx1-2. (F) Mutation of the cebpa promoter to analyze the binding sites of Nkx1-2. (a) Prediction of Nkx1-2 binding sites in cebpa promoter. (b) Analysis of cebpa promoter activity after mutation. (c) The mutation promoter reporter gene expression vector and nkx1.2 expression vector were co-transfected into HEK293T cells, and the luciferase activity was detected. (G) Prokaryotic expression and purification of zebrafish Nkx1-2 protein. M: Marker; lane 1: total Escherichia coli protein before isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction; lane 2: total E. coli protein after IPTG induction; lane 3: protein supernatant; lane 4: inclusion body; lane 5: Nkx1-2 recombinant protein after ultrafiltration; lane 6: purified protein detected by Western blot. (H) Electrophoretic mobility shift assay (EMSA) test to show the binding of Nkx1-2 to cebpa promoter. 1: Negative control; 2: positive control; 3: an excess unlabeled competitor probe; 4: an excess unlabeled competitor probe containing a mutated runt binding site. All data were presented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001; ns means no significant difference). [Color figure can be viewed at wileyonlinelibrary.com]