Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 Page 5 of 17 Figure 4 A: lipid content in adipocytes was quantified with Oil Red O. Absorbance of extracted dye was measured at 500 nm. Data are shown as mean ± SEM (n = 5). Different letters indicate significant differences (p ≤ 0.05). B: superoxide dismutase (SOD) activity in culture media. Different letters indicate significant differences (p ≤ 0.05). Data shown are mean ± SEM (n = 5). adipocytes seem to be able to use alternative means (glyceroneogenesis) to produce glycerol-3-phosphate (G3P) needed for TAG synthesis [28,29]. Two key gly- ceroneogenic enzymes, cytoplasmic malate dehydrogen- ase (MDHC) and cytosolic phosphoenolpyruvate carboxykinase (PEPCKC) were also up-regulated during terminal differentiation. These changes did not necessa- rily mean a switch to anaerobic metabolism. Microarray analyses revealed a decrease of hypoxia inducible factor (HIF1a) while the negative regulator of HIF1a (HIF prolyl hydroxylase) was induced (Fig. 5). Day 15 was marked with the expression changes of genes involved in differentiation and cell cycle. Increase was observed in the anti-osteogenic [30] growth arrest specific protein 6 (GAS6) and adipogenic markers adip- sin and visfatin (Fig. 5). The up-regulations of RBL1, p53-like protein and growth arrest and DNA-damage- inducible (GADD45) g were in concordance with the marked attenuation of cell proliferation. Interestingly, many immune-related genes exhibited sustained up-reg- ulations, which began shortly before or at the onset of terminal differentiation (Fig. 6). Programmed death ligand 1 (PD-L1) is involved in the blockage of cell cycle in T-cells [31]. The up-regulation of decoy receptor interleukin (IL) 13 receptor alpha 2 (IL13Ra2) implies suppression of the anti-inflammatory IL4/IL13 axis [32]. Janus kinase 1 (JAK1) and 5-lipoxygenase activating pro- tein (FLAP) play pivotal roles in respectively, cytokine receptor signalling and biosynthesis of eicosanoids (lipid mediators of inflammation). Galectins (Fig. 5) are carbo- hydrate binding proteins involved in various immune processes [33]. Notably, expression of a panel of immune genes decreased by day 30. The most noticeable aspect of the last stage of adipo- genesis was co-ordinated activation of genes involved in various stress responses. Several components of the acti- vator protein complex 1 (AP1) (c-Fos, c-Jun, JunB1, JunB2, and JunD), which co-ordinates responses to pathogens and stressors, showed highest expression levels at day 30 (Fig. 5). The observed gene expression changes clearly revealed the endoplasmic reticulum (ER) stress in our culture at this stage. The up-regulated oxi- dant stress-activated serine/threonine kinase 25 (YSK1, Fig. 5) is a Golgi complex-associated regulator of trans- port of proteins and lipids to plasma membrane [34]. The A-kinase anchor protein 9 (AKAP9) is an essential mediator in lipolytic pathways and is necessary for the maintenance of Golgi structure through interactions with signalling proteins, including the protein kinase N (PKN1); both AKAP9 and PKN1 showed strong down- regulation at day 15 (Fig. 5). The ER stress response involves a set of mechanisms referred to as unfolded protein response (UPR) [35]. The classical UPR markers X-box-binding protein 1 (XBP1) and activating tran- scription factor 6 (ATF6) were up-regulated during the lipid-loading phase of adipogenesis (Fig. 9). Our findings point to the increased responses to oxidative stress with time. The glutathione peroxidases (GPXs) (Fig. 5 and 9), which are sensitive to lipid mediated peroxidation [36], were similarly induced and a suite of genes coding for proteins of glutathione metabolism also had high levels, at the time when cells engaged in increased LD forma- tion and expansion. The thioredoxin (TXN) antioxidant system is also involved in the regulation of intracellular ROS [37]. The production of ROS is catalyzed with iron and hem, therefore, the up-regulation of ferritin (the intracellular iron storage protein), and two key enzymes in the metabolism of hem, aminolevulinate δ synthetase and heme oxygenase 2 (HMOX2) suggest the induction of mechanisms that protect against oxidative stress at day 30. The massive down-regulation of components of the translational machinery and translation initiation factors suggested reduction of ribosomal biogenesis and attenuation of protein translation while the highest expression level of 18S at day 15 suggested profound changes in the composition of ribosomes during term- inal differentiation (Fig. 5, 9 and 10). Averaged expres- sion profiles of thirty five highly co-expressed ribosomal proteins, components of the 40S and 60S ribosomal sub- units, were shown in Fig. 10. Interestingly, eukaryotic initiation translation factor 5 (eIF5) involved in the