Figure 1

Transactivation of Nnmt by CEBPb is regulated by dexamethasone during the early phase of adipogenesis. (A) Schematic representation of 3T3-L1 pre-adipocyte differentiation protocol and chronological progression through the different phases of the cell cycle during the early phase of adipogenesis. (B) mRNA levels of Nnmt gene measured by Real-Time PCR after post confluent 3T3-L1 cells (T0) were exposed for 48 h to individual components of the DIM alone or in combination. Values are expressed as fold change (mean ± S.D, n = 3) relative to the untreated control (T0). (C) After DEX treatment, mRNA levels of Cebpb, Cebpa and Nnmt were analyzed at the times indicated by RT-qPCR. Data were normalized to the T0 time point of each gene. (D) CEBPB and CEBPA binding sites in the proximal and distal region of Nnmt promoter, obtained from the GTRD database, also including surrounding ENCODE cis- regulatory elements. Black rectangles represent the 8 putative CEBPB binding sites (CBS), corresponding to the amplicons spanning the ChIP-qPCR primer-targeted regions. (E) Effect of DEX on CEBPB recruitment to the Nnmt promoter. Post-confluent 3T3-L1 cells were treated, or not, with DEX (1uM) for 20 h and CEBPB binding on Nnmt promoter was analyzed by Chip-qPCR, with primer pairs specific to the putative CEBPB binding sites. Chip-qPCR was performed using control IgG and anti-CEBPB antibody. Chip data were expressed as percentage of input normalized to control IgG. Statistical significance was calculated by means of a one-sided Welch’s t-test between DEX- and DEX+ conditions for each amplicon. For interpretation purposes, only the regions that showed enrichment with anti-CEBPB higher than the control IgG for the same sets of primers were considered for the final statistical assignments. (F) 3T3-L1 cells were transfected with reporter plasmids containing the full Nnmt promoter (~ 1500 bp) or two mutant constructs (mut CBS4 and mut CBS8) and grown two days post-confluence before treatment, or not, with DEX for 20 h. Firefly and renilla luciferase activity were measured sequentially and the results expressed as ratios of firefly luciferase activity over relative renilla luciferase activity. An empty reporter vector was also used as internal control. The data represent three independent experiments, each with at least three technical replicates, and values are expressed as mean ± SEM. Statistical significance was calculated by means of a one-sided Welch’s t-test. For all the panels: *p < 0.05; **p < 0.01; ***p < 0.001.