Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 3 of 17 D1 D4 D5 D6 D7 D9 D10 D11 D12 D15 D20 D23 D30 Subconfluent Confluent Hormonal induction Terminal differentiation Medium= L-15 FBS (10%) Antibiotics L-glutamin Medium +Insulin +Dex +IBMX +T3 +Biotin +Pantoten Medium +lipid mix Figure 1 Summary of procedures and the key stages. a proinflammatory cytokine and a potent negative regu- lator of adipocyte differentiation in terrestrial verte- brates, was up-regulated before confluence as well as a panel of TNF-related genes and receptors (Fig. 5 and 6). We observed up-regulation of PPARg already at day 4 (Fig. 7). This is the master regulator that co-ordinately activates transcription of adipocyte-specific genes [17,18]. Figure 2 Cell division, DNA replication and nucleotide metabolism. A: cell proliferation was assessed by immunocytochemical detection of proliferating cell nuclear antigen (PCNA). Data (labeling index) are shown as mean ± SEM (n = 5). Different letters indicate significant differences (p ≤ 0.05). B: expression changes (mean log-ER ± SE) of eight genes (Cdc45, cell division control protein 42 homolog, anti-silencing function 1B, ribonucleoside-diphosphate reductase large subunit, cold inducible RNA binding protein-1 and 2, chromosome segregation 1-like protein, chromosome-associated kinesin KIF4A) analyzed with microarrays. FBS..........Fetal Bovine Serum Dex.......... Dexametasone IBMX....... Isobutylmethylxanthine T3.............Triiodothyronine D1,D4,D7,D9,D15,D30 = Microarray & qPCR D5,D6,D10,D11,D12,D20,D23,D30 = qPCR Confluence (day 7) and hormonal induction (day 9) Cells reached confluence at day 7. Up-regulation of reti- noblastoma-like protein 1 (RBL1) and p53-like protein (Fig. 5) was in concordance with the temporary cell cycle arrest at G0/G1, which is commonly observed at this stage in most mammalian cell lines. After stimula- tion with hormones, cells re-enter the cell cycle and undergo mitotic clonal expansion before ultimate exit from the cell cycle. At day 9, expression of RBL1 and p53-like protein decreased while cyclins E1 and B2 were stimulated. Two days after addition of the differentiation media, cells changed to a more rounded shape. This coincided with the up-regulation of a suite of motor contractile proteins (Fig. 5). Interestingly, microarray showed elevated expression of TAGLN at day 7 and day 9. TAGLN is a marker of pericytes, smooth muscle-like cells surrounding vasculature, that are closely related to the MSCs and fibroblasts [19]. Positive and negative reg- ulators of myogenic differentiation showed opposite changes: decrease in myogenic factor D (MyoD) and increase in bone morphogenic protein 4 (BMP4) (Fig.7). We also observed up-regulation of several genes for Ca2 + binding proteins (Fig. 5). Calcium is involved in the control of the whole adipogenic process, from multipo- tent stem cells to adipocytes [20,21]. Increases in cyto- plasmic calcium during this phase inhibit adipogenesis [21-24]. Days 7 and 9 were marked by the expression changes of C/EBPs. The early adipogenic marker C/ EBPb, was up-regulated until confluence, whereas tran- sient induction of C/EBPδ and consistent increase of C/ EBPa were observed after the addition of hormones at day 9 (Fig. 7). Overall, C/EBPb and C/EBPδ work sequentially and predate the expression of C/EBPa [12]. We observed different expression changes in a number of genes encoding chemokines and cytokines. The leu- kocyte cell-derived chemotaxin 2 (LECT2) was re-acti- vated at day 9 and remained up-regulated until the end of the study period (Fig. 6). LECT2 is a potent neutro- phil chemoattractant, which also affects development of