Skip to main content
  • Research article
  • Open access
  • Published:

A transcriptome analysis identifies molecular effectors of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells



The deposition of unconjugated bilirubin (UCB) in selected regions of the brain results in irreversible neuronal damage, or Bilirubin Encephalopathy (BE). Although UCB impairs a large number of cellular functions in other tissues, the basic mechanisms of neurotoxicity have not yet been fully clarified. While cells can accumulate UCB by passive diffusion, cell protection may involve multiple mechanisms including the extrusion of the pigment as well as pro-survival homeostatic responses that are still unknown.


Transcriptome changes induced by UCB exposure in SH-SY5Y neuroblastoma cell line were examined by high density oligonucleotide microarrays. Two-hundred and thirty genes were induced after 24 hours. A Gene Ontology (GO) analysis showed that at least 50 genes were directly involved in the endoplasmic reticulum (ER) stress response. Validation of selected ER stress genes is shown by quantitative RT-PCR. Analysis of XBP1 splicing and DDIT3/CHOP subcellular localization is presented.


These results show for the first time that UCB exposure induces ER stress response as major intracellular homeostasis in surviving neuroblastoma cells in vitro.


The toxic effects of bilirubin, a bile pigment produced during the catabolism of heme-containing compounds [1], have been documented in various biological systems. Nearly all newborn infants develop increased levels of unconjugated bilirubin (UCB) in the blood stream, clinically evident as neonatal jaundice [2]. Although this is a benign and transient phenomenon, abnormal accumulation of bilirubin may cause bilirubin encephalopathy ranging from minimal neurological injury to severe and permanent condition leading to neurodevelopmental dysfunctions [3]. Furthermore, infants affected by the genetic pathology of Crigler-Najjar I present high UCB plasma levels and are particularly exposed to bilirubin encephalopathy (BE) [4].

The basic mechanisms of hyperbilirubinemia neurotoxicity have not been fully clarified, although it has been shown that UCB may impair a large number of cellular functions, including energy metabolism [5], cell proliferation [6], DNA and protein synthesis [7], receptor functionality [8], and neurotransmitter uptake and release [9]. Furthermore, in the brain UCB seems more injurious to neurons than to glial cells, with effects that depend on the cellular subtype [10].

The endoplasmic reticulum (ER) plays a crucial role in the synthesis and folding of newly secretory and membrane proteins [11]. Any perturbation that compromises protein folding and functionality of the ER is referred to as ER stress. The ER stress response can promote cellular repair and survival by reducing the load of unfolded proteins through global attenuation of protein synthesis and by upregulating chaperones [12]. This response is collectively termed as the unfolded protein response (UPR) and is mediated by three ER transmembrane receptors: pancreatic ER kinase PERK (also known as eukaryotic translation initiation factor 2α (eIF2α) kinase 3 (EIF2AK3)), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6).

In resting cells, all of these ER stress receptors are maintained in an active state through their association with the ER chaperone GRP78 (also called BiP). An accumulation of unfolded proteins causes dissociation of GRP78 from PERK, IRE1 and ATF6, thereby initiating the UPR. Thus UPR is a pro-survival response to reduce the accumulation of unfolded proteins and restore normal function [13]. However, when misfolded-protein aggregation persists and ER stress cannot be resolved, signalling switches from a pro-survival to a pro-apoptotic pathway.

Recently, the ER has attracted attention as a key subcellular compartment in which the effects of several cellular stresses may contribute to pathological processes culminating in neuronal injury and degeneration. A central role of ER dysfunction is evident in various pathologies of the brain, including acute injuries (transient ischemia, trauma) and neurodegenerative disorders (Alzheimer's, Parkinson's and Huntington's diseases) [14].

The present study was designed to analyze the consequences of bilirubin exposure to better understand the molecular processes underlying neuronal damage and homeostasis. We used high density oligonucleotide microarrays to analyze the gene expression profile of human neuroblastoma SH-SY5Y cells upon UCB treatment. Gene expression data and experimental validation point to ER stress as the major intracellular homeostatic response in surviving cells.


Characterization of the cell viability of UCB treated SH-SY5Y cells

The human neuroblastoma SH-SY5Y cell line is a well-established in vitro cellular model to study the biological consequences of neuroactive molecules, drugs and toxins. To assess UCB effects, we have taken advantage of previous studies where the outcome of clinically relevant UCB concentrations was tested and the cytotoxic effects revealed [15, 16]. The sensitivity of SH-SY5Y cells to UCB treatment was thus evaluated by measuring cell viability after exposing cells to a concentration of free bilirubin of 140 nM (Bf 140 nM) for 1, 4 and 24 h. As shown in Figure 1, cell viability was reduced to 60% already after 1 h of UCB treatment and the extent of reduction in cell viability never exceeded the 40% of the cell population, even with longer treatments.

Figure 1
figure 1

Cell viability in SH-SY5Y cells treated with UCB. SHSY5Y were exposed to UCB treatment at Bf 140 nM during a time course of 1, 4 and 24 hours and cell viability was evaluated by MTT assay. Cell viability of UCB-treated samples was expressed as a percentage of the respective samples treated with DMSO 0.6%. Data are expressed as the mean ± SD of 3 independent experiments performed in triplicate.

Gene expression analysis by oligonucleotide arrays in UCB treated SH-SY5Y cells

To examine the molecular events associated with the homeostatic response to UCB treatment, high density oligonucleotide microarrays (Affymetrix GeneChip Human U133A 2.0) were used to interrogate the expression of over 14,500 transcripts. RNA was isolated from SH-SY5Y neuroblastoma cells treated with Bf 140 nM for 1, 4 and 24 h. Experiments were performed in three biological replicates with control samples monitoring the effects of DMSO used as Bf solvent.

Analysis of the microarray hybridization data, as reported in Methods, produced a list of 258 probe sets with a false discovery rate (FDR) of 10%. This list corresponded to 230 genes upregulated after 24 h of UCB treatment ("late response"). No down-regulated genes were detected. The fold change of transcript levels ranged from 1.2 to 6.9, the majority of which being toward the lower end. A list of all differentially expressed genes is provided in the Additional file 1. No significant changes of gene expression were observed after 1 and 4 h of UCB treatment ("early response"). We obtained a list of 58 probe sets when a FDR of 0% was applied and a list of 196 probe sets with a FDR of 5%. Furthermore, 400 probe sets were induced with a FDR of 20% with no down-regulated genes (data not shown).

Differentially expressed genes were then examined for their Gene Ontology (GO) annotations concerning their cellular localization. As shown in Table 1 and as detailed in the Additional file 2, the analysis revealed that these genes were mainly distributed in the endoplasmic reticulum (ER), the Golgi apparatus (Golgi) and in the nucleus. To obtain insights into the pathways in which they were mainly involved, genes were sorted into distinct functional subgroups. UCB treatment induced genes belonging to functional categories related to the UPR in the protein secretory pathway (Table 2) as well as to regulation of transcription and DNA-dependent processes (Table 3). A set of up-regulated genes included plasma membrane transporters of the family of the solute carrier transporters (SLCs) and other genes involved in amino acid metabolism (Table 4). In the remaining part of the manuscript the attention will be focused on the UPR and ER stress responses, since the majority of up-regulated genes are well known members of these homeostatic pathways.

Table 1 Identification of the cellular localization of differentially expressed genes in response to 24 h of UCB treatment at Bf 140 nM in SH-SY5Y cells.
Table 2 Differentially expressed genes involved in ER stress signalling and UPR in response to UCB treatment in SH-SY5Y cells.
Table 3 Differential expression of genes mapping to "regulation of transcription, DNA-dependent" (GO Biological Process) in response to UCB treatment in SH-SY5Y cells.
Table 4 Differential expression of genes involved in protein synthesis/translation, amino acid metabolism/transport and autophagy in response to UCB treatment in SH-SY5Y cells.

UCB treatment induced expression of ER stress genes in the UPR of the secretory pathway

UPR serves many aspects of the secretory pathway and is predominantly involved in the response to stress conditions when proteins are misfolded. GO categories of UPR components induced by UCB are shown in Table 2. These include stress sensing and signalling, protein folding, translocation, vesicular transport and ER-associated degradation. Genes known or predicted by sequence homology to play a physiological role in secretion or in the biogenesis of organelles were also increased.

The largest group of up-regulated genes was represented by ER-resident molecular chaperone and oxido-reductase proteins. Among the molecular chaperones, we found established UPR target genes such as HSPA5 (heat shock 70 kDa protein 5) (upregulated 2.3 times), known also as GRP78 (glucose-regulated protein, 78 kDa) or BiP [17], HSP90B (heat shock protein 90 kDa beta (Grp94) (1.5), member 1), DNAJB9 (4.4) and DNAJC10 (1.8)[18]. Additional genes included PDIA5 (1.7), PDIA6 (1.5) and ERO1LB (2.1) that are involved in oxidative protein folding [19].

A second major group was composed by genes involved in co-translational translocation of proteins across the ER membrane, to the Golgi and in the anterograde/retrograde transport back into the cytosol. The group included the signal sequence receptor SSR1 (1.6), the translocating chain-associating membrane protein TRAM1 (1.8), members of the SEC61 complex, such as SEC61A1 (1.6), SEC61A2 (1.4) and SEC63 (1.5), and SERP1 (Stress-associated Endoplasmic Reticulum Protein 1) (1.9), also known as RAMP4 (Ribosome-Associated Membrane Protein 4), that is a Sec61-associated polypeptide specifically induced by ER stress [20]. Up-regulated genes were also represented by the coated vesicle membrane proteins, such as SEC23B (1.8), SEC24D (2.1), SEC31A (1.6) and SCFD1 (1.5), as well as by the transmembrane trafficking proteins TMED2 (1.4), TMED9 (1.6) and TMED10 (1.4).

The ER-associated degradation (ERAD) system eliminates misfolded proteins by their retrotranslocation across the ER membrane into the cytosol, where ubiquitin-conjugating enzymes target them for proteosomal degradation. ERAD requires a number of dedicated ER-resident factors. Indeed, HERPUD1 or HERP (Homocysteine-induced ER Protein) (4.1) [21] was significantly induced as well as EDEM1 (ER Degradation Enhancer Mannosidase alpha-like 1) (2.1) [22], SEL1L (2.1), a transmembrane cofactor of the E3 ubiquitin ligase HRD1 [23], and DERL2 (Derlin membrane-like domain family member 2) (1.6) whose product associates with EDEM1 and is a specific target of the IRE1 branch of the UPR response [24].

UCB treatment induced expression of proteins relevant in the regulation of transcription during ER stress response

As shown in Table 3, several differentially expressed genes were classified into the GO category of "regulation of transcription, DNA-dependent". Up-regulated transcription factors included two established UPR target genes: DDIT3 (6.7), also known as CHOP or C/EBP homologous protein, and XBP1, X box-binding protein 1 (2.6). CHOP is a 29-kDa leucine zipper transcription factor that is ubiquitously expressed at a low level and robustly up-regulated in response to various stress conditions [25]. CHOP is proapoptotic and a key mediator of ER stress-induced cell death [26, 27]. XBP1 gene, after a nonconventional splicing event [28], codes for a transcription activator that regulates a subset of ER-resident chaperones that are essential for protein folding, maturation and degradation in the ER [29]. Additional stress-responsive transcriptional regulators induced by UCB included ATF3 (4.9), ATF4 (1.7), ATF5 (1.6), CEBPB (4.9), CEBPG (2.4), JUN (3.5) and JUND (1.6). Interestingly, although ATF4 showed a moderate induction, several of its downstream targets were strongly up-regulated, e.g. DDIT3, ATF3, CEBPB, CEBPG, CREB3L2 (1.7) and TRIB3 (6.7). TRIB3 (also known as NIPK, SKIP3, TRB3 and SINK) is an ER stress-inducible gene that is involved in CHOP-dependent cell death as a second messenger during ER stress [30].

UCB treatment induced expression of genes involved in protein synthesis/translation, autophagy and aminoacid transport/metabolism

The translational machinery for a new boost of protein synthesis has been suggested to be amplified during UPR-associated translational attenuation [13, 31]. As shown in Table 4, we observed an up-regulation of 6 tRNA synthetase genes, such as AARS (1.7), CARS (1.8), GARS (1.8), MARS (1.5), SARS (2.2) and WARS (2.9), which are involved in tRNA processing. We also observed the induction of 2 eukaryotic translation initiation factors, that are EIF1 (1.4) and EIF5 (1.6). One of the stronger up-regulated genes was FAM129A (family with sequence similarity 129, member A) (6.9), also known as NIBAN, which has been recently demonstrated to be involved in the ER stress response and to positively affect the protein translation machinery by regulating the mTOR pathway [32]. Interestingly, the list of genes induced during UCB treatment also includes DDIT4 (DNA-Damage-Inducible Transcript 4) (4.4), also known as REDD1/RTP801/Dig2, that is another well-known regulator of the mTOR pathway [33]. The mTOR-dependent pathway is strongly connected to the autophagic machinery, and indeed autophagy-related genes such as GABARAPL1 (GABA(A) receptor-associated protein like 1 or ATG8) (1.9), GABARAPL3 (GABA(A) receptors-associated protein like 3 (pseudogene)) (2.3) and WIPI1 (WD repeat domain, phosphoinositide interacting 1 gene or Atg18) (2.8) were also positively differentially expressed, suggesting that surviving neuroblastoma cells may present an induction of autophagy [34, 35].

UCB exposure also activated genes involved in amino acid metabolism (Table 4). Among the strongest up-regulated genes, SLC7A11 (6.8) and SLC3A2 (3.8) were identified. These two genes encode respectively for xCT, the light chain subunit, and for 4F2hC, the glycosylated heavy-chain subunit, that are components of the system xCT, anionic amino acid transport system specific for cystine and glutamate [36]. Recently, the UCB-induced SLC7A5 (3.2) has been shown to physically and functionally interact with SLC3A2, forming a bidirectional amino acid transporter and representing a key regulator of both the mTOR pathway and autophagy [37].

Validation of selected genes by quantitative RT-PCR (qRT-PCR)

For validation, we selected genes considered of interest based on their involvement in ER stress response. Gene expression levels of DDIT3, HSPA5, XBP1, HERPUD1, TRIB3, FAM129A and SLC7A11 were verified by qRT-PCR. Results are reported in Figure 2. The fold differences determined by qRT-PCR were very similar to those obtained by the high density oligonucleotide microarray gene expression analysis.

Figure 2
figure 2

Analysis of gene expression related to the ER stress response. Gene expression levels of selected genes (X axis) were analyzed by qRT-PCR. Y axis represents the gene expression level normalized to housekeeping genes (GAPDH and β-actin) of SH-SY5Y cells not treated (untreated) or treated with vehicle (DMSO 0.6%) or UCB (Bf 140 nM) for 24 hours. These results represent at least three RNA samples per experimental condition run in triplicate.

Analysis of XBP1 splicing and DDIT3/CHOP subcellular localization in UCB treated SH-SY5Y cells

We investigated two independent events that are known activators of the ER stress transcriptional program: unconventional splicing of XBP1 and cytoplasm/nuclear shuttling of DDIT3/CHOP. The splicing of XBP1 transcript changes its open reading frame to code for a transcription factor (spliced XBP1 or sXBP1) that activates the expression of a subset of ER stress/UPR related genes [28, 29]. Here we show that UCB treatment is indeed able to trigger this unconventional splicing (Figure 3A). The nuclear localization of the transcription factor DDIT3/CHOP [25] is one of the most commonly used indicators of ER stress. As presented in Figure 3B, we observed a diffuse cytoplasmic localization in untreated and DMSO 0.6% control cells, but a major nuclear staining in cells treated with Bf 140 nM. This indicates activation of a CHOP-dependent transcriptional program. These data represent an independent proof of the induction of a vigorous ER stress.

Figure 3
figure 3

Analysis of XBP1 splicing and DDIT3/CHOP subcellular localization in SH-SY5Y cells treated with UCB. (A) mRNA levels of the spliced form of XBP1 (s - spliced, u - unspliced), indicator of ER stress, were examined by RT-PCR after 24 h of treatment. The amplified products were separated on a 2% agarose gel and visualized with ethidium bromide staining. Data are representative of n ≥ 2 separate experiments per condition point. (B) The expression of CHOP (shown in red) was examined with immunocytochemistry as described in Methods. Representative images are shown for SH-SY5Y cells not treated (untreated) or treated with vehicle (DMSO 0.6%) or UCB (Bf 140 nM). The nuclei (shown in blue) were visualized with DAPI staining. Bar = 10 μm.


Bilirubin encephalopathy is one of the consequences of severe hyperbilirubinemia and is characterized by multifocal deposition of UCB in selected regions of the brain, resulting in temporary or permanent impairment of auditory, motor or mental functions [38]. Cellular UCB level is closely related to the free concentration (Bf) of the pigment in plasma and to the mechanisms regulating the traffic of UCB among plasma, cerebral spinal fluid and cells. Although UCB is formed in virtually all cells and may enter by passive diffusion [39], intracellular concentration is determined by cellular export and metabolic transformation of the pigment. Interestingly, only certain neuronal cell types present a UCB susceptibility to undergo necrosis and apoptosis [40], whereas astrocytes are relatively spared. This has been linked to a higher content of MRP1, a transporter with high affinity to UCB [41] able to keep the intracellular UCB concentration low. Importantly, the level of MRP1 expression in human neuroblastoma SH-SY5Y cells is inversely and linearly correlated with UCB toxic effects [16].

The molecular mechanisms of intracellular UCB toxicity are still unclear [42]. UCB has been initially proved to be a pro-apoptotic agent, suppressing cell growth by inducing DNA fragmentation, mitochondrial release of cytochrome c, activation of caspase-3 and cleavage of poly(ADP)ribose polymerase [40]. More recently, oxidative stress has emerged as a potential crucial event, since its generation mirrors UCB-mediated apoptosis [43, 44]. In various cellular systems, UCB causes reactive oxygen species (ROS) production, protein oxidation, lipid peroxidation and disruption of glutathione metabolism [43, 4547]. Furthermore, UCB-mediated oxidative stress is also in part responsible for inhibition of cell growth [48, 49]. Inhibition of cell proliferation has been indeed observed in primary vascular smooth muscle cells in vitro as well as inferred by gene expression data on the repression of cell cycle-related genes in the liver of a mouse model of Crigler-Najjar type I disease [50]. To study the effects of intracellular UCB on neurons, we took advantage of the human neuroblastoma SH-SY5Y cell line. These cells have been widely used in neurobiology as the in vitro system of choice to dissect molecular pathways leading to neurodegeneration. Previous studies have already shown that neuroblastoma cells are a good in vitro model to address the consequences of clinically relevant UCB concentrations [16]. Although we are aware of the limitations in the use of a neuroblastoma cell line to recapitulate dysfunctions that occur in neonatal human brain, this experimental model allowed us to dissect in details the molecular events elicited by UCB.

Treatment with Bf 140 nM was highly toxic, triggering death in 40% of the cells within one hour. This is expected since the accepted threshold for bilirubin toxicity approximately occurs at 70 nM [5]. 63% reduction in cell viability at 4 h after bilirubin exposure were previously demonstrated in the rat neuroblastoma N-115 cell line [7]. Furthermore, Silva RF et al. [51] showed 85 nM Bf triggered deleterious effects on mitochondrial function.

A full understanding of molecular mechanisms of cell death will require additional investigation. UCB has been previously proved to induce apoptosis in neuroblastoma cells, although necrosis could not be excluded [52].

Furthermore, the criteria to distinguish sensitive from insensitive neuroblastoma cells remain unclear. One may speculate that subtle heterogeneity in the level of differentiation may play a role in susceptibility to insults. Alternatively, cell death induction may depend on cell cycle position.

We then focused on the 60% of surviving cells to test the hypothesis that these neurons may express a homeostatic response.

A gene expression approach was carried out to identify gene expression patterns and signalling pathways induced upon UCB treatment. No significant changes of gene expression were observed after 1 and 4 h of UCB treatment. After 24 h, 230 genes were induced while no down-regulated genes were observed even when an FDR of 20% was applied.

The lack of an "early response" is surprising since it fails to provide a potential mechanism for cell survival within the first hour. Therefore, present data suggest that it is very unlikely that the molecular pathways induced at 24 hours play a role in the survival of cells after one hour of treatment.

GO analysis of the "late response" genes proved for the first time that UCB treatment induces an ER stress response in neuroblastoma cells in vitro. Evidences for ER stress involvement are overwhelming. Among others, genes induced by UCB treatment include several molecular chaperones like BiP, a well-known marker of ER overload, as well as molecular components of the ERAD system, that has been recently positioned in a central stage among the molecular mechanisms of neurodegeneration [53, 54]. Furthermore, two well-known events in ER stress, such as XBP1 unconventional splicing and DDIT3/CHOP nuclear relocation, were proved to be triggered by UCB treatment, therefore providing additional experimental evidences.

UCB also altered several components of the translational machinery including tRNA synthesis genes and translation initiation factors. The translation machinery is considered one of the major targets of the ER stress response to regulate protein synthesis and decrease protein overload. Since no data are available in literature about the relationship between UCB intracellular concentration and translation rate, there is a compelling need for an accurate proteomic analysis of UCB effects in SH-SY5Y neuroblastoma cells. A complementary cellular strategy in ER stress conditions includes an increase in protein degradation. In this context some UCB-induced genes suggested a role for autophagy. It is of notice that autophagy has been recently associated with neurodegenerative diseases as well as with inhibition of cell growth and entering into a quiescent state [55, 56]. This may correlate to previous reports showing a severe inhibition of cell proliferation mediated by UCB [49]. Further experimental validation is needed to prove an increase of autophagy.

Finally, the induction of both subunits of the transport system X(C)(-) was also observed: SLC3A2 (4F2hc) and SLC7A11 (xCT) are able to heterodimerize to mediate cystine-glutamate exchange and regulate intracellular glutathione levels. Since the maintenance of a high glutathione concentration may be protective [57], we speculate that induction of the X(C)(-) transport system may represent a major pro-survival response, as has been recently shown in neurons [58]. Interestingly, we could not observe any significant differential gene expression of MRP1 at any point of the time course suggesting that this transporter plays a negligible role in UCB-induced adaptation in this in vitro model.


In summary, the present study shows for the first time that intracellular accumulation of UCB in surviving neuroblastoma SH-SY5Y cell line provokes ER stress.



UCB was purchased from Sigma (St. Louis, MO, USA) and purified as described by McDonagh and Assisi [59]. UCB was dissolved in 0.6% v/v of DMSO and diluted in 100 μL of complete medium supplemented with 15% Fetal Bovine Serum containing 54 μM albumin. Bf concentration was 140 nM, measured as described previously [60]. 3(4,5-dimethylthiazolyl-2)-2,5 diphenyl tetrazolium (MTT), HPLC-grade dimethyl sulfoxide (DMSO), and all other chemicals were purchased from Sigma, unless otherwise specified.

Experimental treatment of SH-SY5Y cell line

SH-SY5Y human neuroblastoma cells (ATCC CRL-2266) were cultured in a humidified incubator at 37°C and 5% CO2 in F12-EMEM (1:1) containing 15% foetal bovine serum (FBS), 2 mM L-glutamine, 25 μg/mL gentamicin, 100 U/ml penicillin, and 100 μg/mL streptomycin at 37°C with 5% CO2. Cells were seeded at 6 × 106 cells in 75 cm2 flasks and grown for 24 h (cells reached 70-80% confluence). The medium was then replaced with experimental medium, which was either free medium (untreated samples) or 140 nM Bf medium (Bf 140 nM samples), or 0.6% DMSO medium (DMSO 0.6% samples) and cells were allowed to grow. After 1, 4 and 24 h cells were collected and processed for cell viability (MTT cell viability assay), microarray, quantitative RT-PCR and immunofluorescence analyses. Separate flasks of cells were used for each of the treatments and assays. Experiments were performed at least in three independent biological replicates at all time points.

MTT cell viability assay

Cells were treated as described above and the MTT cell viability assay was performed as previously described by Denizot and Lang [61]. Absorbance was measured at 562 nm using a microtiter plate reader (LC 400, Beckman Coulter, Milan, Italy). Cell viability of UCB treated samples was expressed as a percentage of the respective samples treated with DMSO alone.

RNA extraction and quantitative RT-PCR (qRT-PCR)

Total RNA was isolated using the TRIzol reagent (Invitrogen) following the manufacturer's instructions. Single strand cDNA was obtained from 1 μg of purified RNA using the iSCRIPT™ cDNA Synthesis Kit (Bio-Rad) according to manufacturer's instructions. Quantitative RT-PCR was performed using SYBR-Green PCR Master Mix (Applied Biosystem) and an iCycler IQ Real Time PCR System (Bio-Rad). Sequence of gene specific primers used for qRT-PCR is provided in Table 5. Expression of the gene of interest was normalized to house keeping genes (glyceraldehyde-3-phosphate dehydrogenase or GAPDH and β-actin) and the initial amount of the template of each sample was determined as relative expression versus one of the samples chosen as reference. The relative expression of each sample was calculated by the formula 2 exp-ΔΔCt (User Bulletin 2 of the ABI Prism 7700 Sequence Detection System). GAPDH and β-actin expressions are not modified under the present experimental conditions (data not shown). XBP1 splicing was studied using reverse transcriptase-polymerase chain reaction. cDNA was synthesized as herein reported. Human XBP1 primers (forward: 5'-AAACAGAGTAGCAGCTCAGACTGC-3'; reverse: 5'-TCCTTCTGGGTAGACCTCTGGG AG-3') were designed to generate cDNA products encompassing the IRE1 cleavage site as previously described [28]. The unspliced (uXBP1) and spliced (sXBP1) mRNAs generate 473- and 447-bp cDNA products, respectively. The amplified products were separated on a 2% agarose gel and visualized with ethidium bromide staining.

Table 5 Primer sequences used for qRT-PCR.

Microarray processing and data analysis

Total RNA was extracted as described above and purified using the RNeasy mini kit (Qiagen). The quality of total RNA was assessed using a bioanalyzer (Agilent 2100; Agilent Technologies) and RNA was quantified by using a ND-1000 Nanodrop spectrophotometer. Ten μg of each total RNA sample was labelled according to the standard one-cycle amplification and labelling protocol developed by Affymetrix (Santa Clara, CA). Labelled cRNA was hybridized on Affymetrix GeneChip Human U133A 2.0 Arrays containing over 14,500 transcripts. Hybridized GeneChips were stained, washed (GeneChip Fluidic Station 450) and scanned (GeneChip Scanner 3000 7G). Cell intensity values and probe detection calls were computed from the raw array data using the Affymetrix GeneChip Operating Software (GCOS). Further data processing was performed in the R computing environment, version R 2.5.0 for Windows) using packages from the BioConductor software project Variance-stabilizing normalization was applied [62], using the "justvsn" function from the "vsn" library. Normalized data were then filtered based on the Affymetrix detection call, so that only probes that had a Present call in at least one of the arrays were retained [63]. Data were then imported in the MultiExperiment Viewer (MeV) software [64] (version 4.0.01 for Windows XP), and statistical analysis was performed with the SAM (Significance Analysis of Microarrays) module [65, 66]. A False Discovery Rate (FDR) of about 10% (i.e. 9.317%) was applied to detect significantly differentially expressed genes. Microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) with Accession Number GSE16768. Differentially expressed genes were then specifically examined, based on their Gene Ontology annotation [67], for their cellular localization and for their involvement in some biological processes of interest. GO slim annotations were based on the Generic GO Term Mapper using the Gene Association File goa_human_hgnc (Generic GO slim).


For immunofluorescence experiments, SH-SY5Y cells were cultured on glass slides overnight, fixed in 4% paraformaldehyde for 10 minutes, washed with PBS two times, treated with 0.1 M glycine for 5 minutes in PBS and permeabilized with 0.1% Triton X-100 in PBS for additional 5 minutes. After washing with PBS and blocking with 0.2% BSA in PBS, cells were incubated with the anti-CHOP 1:100 (Santa Cruz Biotechnology) diluted in 0.2% BSA in PBS for 90 minutes at room temperature. After washing, cells were incubated with AlexaFluor 594 (Dako Cytomation)-labelled anti-mouse secondary antibody for 1 h. For nuclear staining, cells were incubated with 1 μg/mL DAPI for 5 minutes. Cells were washed and mounted with Vectashield mounting medium (Vector). Images were collected using a confocal microscope (LEICA TCS SP2).


  1. Ostrow JD, Mukerjee P, Tiribelli C: Structure and binding of unconjugated bilirubin: relevance for physiological and pathophysiological function. J Lipid Res. 1994, 35: 1715-1737.

    CAS  PubMed  Google Scholar 

  2. Gourley GR: Bilirubin metabolism and kernicterus. Adv Pediatr. 1997, 44: 173-229.

    CAS  PubMed  Google Scholar 

  3. Cashore WJ: The neurotoxicity of bilirubin. Clin Perinatol. 1990, 17: 437-448.

    CAS  PubMed  Google Scholar 

  4. Seppen J, Bosma PJ, Goldhoorn BG, Bakker CT, Chowdhury JR, Chowdhury NR, Jansen PL, Oude Elferink RP: Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase. J Clin Invest. 1994, 94: 2385-2391. 10.1172/JCI117604.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Roger C, Koziel V, Vert P, Nehlig A: Effects of bilirubin infusion on local cerebral glucose utilization in the immature rat. Dev Brain Res. 1993, 76: 115-130. 10.1016/0165-3806(93)90129-X.

    Article  CAS  Google Scholar 

  6. Keshavan P, Schwemberger SJ, Smith DL, Babcock GF, Zucker SD: Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization. Int J Cancer. 2004, 112: 433-445. 10.1002/ijc.20418.

    Article  CAS  PubMed  Google Scholar 

  7. Kashiwamata S, Aono S, Semba RK: Characteristic changes of cerebellar proteins associated with cerebellar hypoplasia in jaundiced Gunn rat and the prevention of these by phototherapy. Experientia. 1980, 36: 1143-1144. 10.1007/BF01976089.

    Article  CAS  PubMed  Google Scholar 

  8. Hoffman DJ, Zanelli SA, Kubin J, Mishra OP, Delivoria-Papadopoulos M: The in vivo effect of bilirubin on the N-methyl-D-aspartate receptor/ion channel complex in the brains of newborn piglets. Pediatr Res. 1996, 40: 804-808. 10.1203/00006450-199612000-00005.

    Article  CAS  PubMed  Google Scholar 

  9. Ochoa EL, Wennberg RP, An Y, Tandon T, Takashima T, Nguyen T, Chui A: Interactions of bilirubin with isolated presynaptic nerve terminals: functional effects on the uptake and release of neurotransmitters. Cell Mol Neurobiol. 1993, 13: 69-86. 10.1007/BF00712990.

    Article  CAS  PubMed  Google Scholar 

  10. Notter MFD, Kendig JW: Differential sensitivity of neural cells to bilirubin toxicity. Exp Neurol. 1986, 94: 670-682. 10.1016/0014-4886(86)90246-3.

    Article  CAS  PubMed  Google Scholar 

  11. Ron D, Walter P: Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 2007, 8: 519-529. 10.1038/nrm2199.

    Article  CAS  PubMed  Google Scholar 

  12. Kaufman RJ: Orchestrating the unfolded protein response in health and disease. J Clin Invest. 2002, 110: 1389-98.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Schröder M, Kaufman RJ: The mammalian unfolded protein response. Annu Rev Biochem. 2005, 74: 739-89. 10.1146/annurev.biochem.73.011303.074134.

    Article  PubMed  Google Scholar 

  14. Scheper W, Hoozemans JJ: Endoplasmic reticulum protein quality control in neurodegenerative disease: the good, the bad and the therapy. Curr Med Chem. 2009, 16: 615-626. 10.2174/092986709787458506.

    Article  CAS  PubMed  Google Scholar 

  15. Calligaris SD, Bellarosa C, Giraudi P, Wennberg RP, Ostrow JD, Tiribelli C: Cytotoxicity is predicted by unbound and not total bilirubin concentration. Pediatr Res. 2007, 62: 576-580. 10.1203/PDR.0b013e3181568c94.

    Article  CAS  PubMed  Google Scholar 

  16. Corich L, Aranda A, Carrassa L, Bellarosa C, Ostrow JD, Tiribelli C: The cytotoxic effect of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells is modulated by the expression level of MRP1 but not MDR1. Biochem J. 2009, 417: 305-312. 10.1042/BJ20080918.

    Article  CAS  PubMed  Google Scholar 

  17. Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D: Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol. 2000, 2: 326-332. 10.1038/35014014.

    Article  CAS  PubMed  Google Scholar 

  18. Ushioda R, Hoseki J, Araki K, Jansen G, Thomas DY, Nagata K: ERdj5 is required as a disulfide reductase for degradation of misfolded proteins in the ER. Science. 2008, 321: 569-572. 10.1126/science.1159293.

    Article  CAS  PubMed  Google Scholar 

  19. Ellgaard L, Helenius A: Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol. 2003, 4: 181-191. 10.1038/nrm1052.

    Article  CAS  PubMed  Google Scholar 

  20. Hori O, Miyazaki M, Tamatani T, Ozawa K, Takano K, Okabe M, Ikawa M, Hartmann E, Mai P, Stern DM, Kitao Y, Ogawa S: Deletion of SERP1/RAMP4, a component of the endoplasmic reticulum (ER) translocation sites, leads to ER stress. Mol Cell Biol. 2006, 26: 4257-4267. 10.1128/MCB.02055-05.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Kokame K, Agarwala KL, Kato H, Miyata T: Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress. J Biol Chem. 2000, 275: 32846-32853. 10.1074/jbc.M002063200.

    Article  CAS  PubMed  Google Scholar 

  22. Oda Y, Hosokawa N, Wada I, Nagata K: EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin. Science. 2003, 299: 1394-1397. 10.1126/science.1079181.

    Article  CAS  PubMed  Google Scholar 

  23. Neuber O, Jarosch E, Volkwein C, Walter J, Sommer T: Ubx2 links the Cdc48 complex to ER-associated protein degradation. Nat Cell Biol. 2005, 7: 993-998. 10.1038/ncb1298.

    Article  CAS  PubMed  Google Scholar 

  24. Oda Y, Okada T, Yoshida H, Kaufman RJ, Nagata K, Mori K: Derlin-2 and Derlin-3 are regulated by the mammalian unfolded protein response and are required for ER-associated degradation. J Cell Biol. 2006, 172: 383-393. 10.1083/jcb.200507057.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Wang XZ, Lawson B, Brewer JW, Zinszner H, Sanjay A, Mi LJ, Boorstein R, Kreibich G, Hendershot LM, Ron D: Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153). Mol Cell Biol. 1996, 16: 4273-4280.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Friedman AD: GADD153/CHOP, a DNA damage-inducible protein, reduced CAAT/enhancer binding protein activities and increased apoptosis in 32D c13 myeloid cells. Cancer Res. 1996, 56: 3250-3256.

    CAS  PubMed  Google Scholar 

  27. Oyadomari S, Mori M: Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004, 11: 381-389. 10.1038/sj.cdd.4401373.

    Article  CAS  PubMed  Google Scholar 

  28. Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, Clark SG, Ron D: IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature. 2002, 415: 92-96. 10.1038/415092a.

    Article  CAS  PubMed  Google Scholar 

  29. Lee AH, Iwakoshi NN, Glimcher LH: XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol. 2003, 23: 7448-7459. 10.1128/MCB.23.21.7448-7459.2003.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Ohoka N, Yoshii S, Hattori T, Onozaki K, Hayashi H: TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death. EMBO J. 2005, 24: 1243-1255. 10.1038/sj.emboj.7600596.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Okada T, Yoshida H, Akazawa R, Negishi M, Mori K: Distinct roles of ATF6 and PERK in transcription during the mammalian unfolded protein response. Biochem J. 2002, 366: 585-594. 10.1042/BJ20020391.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Sun GD, Kobayashi T, Abe M, Tada N, Adachi H, Shiota A, Totsuka Y, Hino O: The endoplasmic reticulum stress-inducible protein Niban regulates eIF2alpha and S6K1/4E-BP1 phosphorylation. Biochem Biophys Res Commun. 2007, 360: 181-187. 10.1016/j.bbrc.2007.06.021.

    Article  CAS  PubMed  Google Scholar 

  33. Kimball SR, Do AN, Kutzler L, Cavener DR, Jefferson LS: Rapid turnover of the mTOR complex 1 (mTORC1) repressor REDD1 and activation of mTORC1 signalling following inhibition of protein synthesis. J Biol Chem. 2008, 283: 3465-3475. 10.1074/jbc.M706643200.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Geng J, Klionsky DJ: The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. EMBO Rep. 2008, 9: 859-864. 10.1038/embor.2008.163.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Proikas-Cezanne T, Waddell S, Gaugel A, Frickey T, Lupas A, Nordheim A: WIPI-1alpha (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy. Oncogene. 2004, 23: 9314-9325. 10.1038/sj.onc.1208331.

    Article  CAS  PubMed  Google Scholar 

  36. Sato H, Tamba M, Ishii T, Bannai S: Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins. J Biol Chem. 274: 11455-11458. 10.1074/jbc.274.17.11455.

  37. Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B, Yang H, Hild M, Kung C, Wilson C, Myer VE, MacKeigan JP, Porter JA, Wang YK, Cantley LC, Finan PM, Murphy LO: Bidirectional transport of amino acids regulates mTOR and autophagy. Cell. 2009, 136: 521-534. 10.1016/j.cell.2008.11.044.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Gourley GR: Bilirubin metabolism and kernicterus. Adv Pediatr. 1997, 44: 173-229.

    CAS  PubMed  Google Scholar 

  39. Zucker SD, Goessling W, Hoppin AG: Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes. J Biol Chem. 1999, 274: 10852-10862. 10.1074/jbc.274.16.10852.

    Article  CAS  PubMed  Google Scholar 

  40. Rodrigues CM, Sola S, Brites D: Bilirubin induces apoptosis via the mitochondrial pathway in developing rat brain neurons. Hepatology. 2002, 35: 1186-1195. 10.1053/jhep.2002.32967.

    Article  CAS  PubMed  Google Scholar 

  41. Gennuso F, Fernetti C, Tirolo C, Testa N, L'Episcopo F, Caniglia S, Morale MC, Ostrow JD, Pascolo L, Tiribelli C, Marchetti B: Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistance-associated protein 1 (Mrp1). Proc Natl Acad Sci USA. 2004, 101: 2470-2475. 10.1073/pnas.0308452100.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. Ostrow JD, Pascolo L, Brites D, Tiribelli C: Molecular basis of bilirubin-induced neurotoxicity. Trends Mol Med. 2004, 10: 65-70. 10.1016/j.molmed.2003.12.003.

    Article  CAS  PubMed  Google Scholar 

  43. Oakes GH, Bend JR: Early steps in bilirubin-mediated apoptosis in murine hepatoma (Hepa 1c1c7) cells are characterized by aryl hydrocarbon receptor-independent oxidative stress and activation of the mitochondrial pathway. J Biochem Mol Toxicol. 2005, 19: 244-55. 10.1002/jbt.20086.

    Article  CAS  PubMed  Google Scholar 

  44. Vitek L, Schwertner HA: The heme catabolic pathway and its protective effects on oxidative stress-mediated diseases. Adv Clin Chem. 2007, 43: 1-57. full_text.

    Article  CAS  PubMed  Google Scholar 

  45. Kumar S, Guha M, Choubey V, Maity P, Srivastava K, Puri SK, Bandyopadhyay U: Bilirubin inhibits Plasmodium falciparum growth through the generation of reactive oxygen species. Free Radic Biol Med. 2008, 44: 602-613. 10.1016/j.freeradbiomed.2007.10.057.

    Article  CAS  PubMed  Google Scholar 

  46. Seubert JM, Darmon AJ, El-Kadi AO, D'Souza SJ, Bend JR: Apoptosis in murine hepatoma hepa 1c1c7 wild-type, C12, and C4 cells mediated by bilirubin. Mol Pharmacol. 2002, 62: 257-264. 10.1124/mol.62.2.257.

    Article  CAS  PubMed  Google Scholar 

  47. Cesaratto L, Calligaris SD, Vascotto C, Deganuto M, Bellarosa C, Quadrifoglio F, Ostrow JD, Tiribelli C, Tell G: Bilirubin-induced cell toxicity involves PTEN activation through an APE1/Ref-1-dependent pathway. J Mol Med. 2007, 85: 1099-112. 10.1007/s00109-007-0204-3.

    Article  CAS  PubMed  Google Scholar 

  48. Ollinger R, Bilban M, Erat A, Froio A, McDaid J, Tyagi S, Csizmadia E, Graça-Souza AV, Liloia A, Soares MP, Otterbein LE, Usheva A, Yamashita K, Bach FH: Bilirubin: a natural inhibitor of vascular smooth muscle cell proliferation. Circulation. 2005, 112: 1030-1039. 10.1161/CIRCULATIONAHA.104.528802.

    Article  PubMed  Google Scholar 

  49. Ollinger R, Kogler P, Troppmair J, Hermann M, Wurm M, Drasche A, Königsrainer I, Amberger A, Weiss H, Ofner D, Bach FH, Margreiter : Bilirubin inhibits tumor cell growth via activation of ERK. Cell Cycle. 2007, 6: 3078-3085.

    Article  PubMed  Google Scholar 

  50. Nguyen N, Bonzo JA, Chen S, Chouinard S, Kelner MJ, Hardiman G, Bélanger A, Tukey RH: Disruption of the ugt1 locus in mice resembles human Crigler-Najjar type I disease. J Biol Chem. 2008, 283: 7901-7911. 10.1074/jbc.M709244200.

    Article  CAS  PubMed  Google Scholar 

  51. Silva RF, Rodrigues CM, Brites D: Bilirubin-induced apoptosis in cultured rat neural cells is aggravated by chenodeoxycholic acid but prevented by ursodeoxycholic acid. J Hepatol. 2001, 34: 402-408. 10.1016/S0168-8278(01)00015-0.

    Article  CAS  PubMed  Google Scholar 

  52. Han Z, Hu P, Ni D: Bilirubin induced apoptosis of human neuroblastoma cell line SH-SY5Y and affected the mitochondrial membrane potential. Zhonghua Er Bi Yan Hou Ke Za Zhi. 2002, 37: 243-246.

    PubMed  Google Scholar 

  53. Paschen W, Mengesdorf T: Endoplasmic reticulum stress response and neurodegeneration. Cell Calcium. 2005, 38: 409-415. 10.1016/j.ceca.2005.06.019.

    Article  CAS  PubMed  Google Scholar 

  54. Vembar SS, Brodsky JL: One step at a time: endoplasmic reticulum-associated degradation. Nat Rev Mol Cell Biol. 2008, 9: 944-957. 10.1038/nrm2546.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  55. Matus S, Lisbona F, Torres M, León C, Thielen P, Hetz C: The stress rheostat: an interplay between the unfolded protein response (UPR) and autophagy in neurodegeneration. Curr Mol Med. 2008, 8: 157-172. 10.2174/156652408784221324.

    Article  CAS  PubMed  Google Scholar 

  56. Rubinsztein DC: The roles of intracellular protein-degradation pathways in neurodegeneration. Nature. 2006, 443: 780-786. 10.1038/nature05291.

    Article  CAS  PubMed  Google Scholar 

  57. Sedlak TW, Saleh M, Higginson DS, Paul BD, Juluri KR, Snyder SH: Bilirubin and glutathione have complementary antioxidant and cytoprotective roles. Proc Natl Acad Sci USA. 2009, 106: 5171-5176. 10.1073/pnas.0813132106.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  58. Savaskan NE, Heckel A, Hahnen E, Engelhorn T, Doerfler A, Ganslandt O, Nimsky C, Buchfelder M, Eyüpoglu IY: Small interfering RNA-mediated xCT silencing in gliomas inhibits neurodegeneration and alleviates brain edema. Nat Med. 2008, 14: 629-632. 10.1038/nm1772.

    Article  CAS  PubMed  Google Scholar 

  59. McDonagh AF, Assisi F: The ready isomerization of bilirubin IX- in aqueous solution. Biochem J. 1972, 129: 797-800.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  60. Roca L, Calligaris S, Wennberg RP, Ahlfors CE, Malik SG, Ostrow JD, Tiribelli C: Factors affecting the binding of bilirubin to serum albumins: validation and application of the peroxidase method. Pediatr Res. 2006, 60: 724-728. 10.1203/01.pdr.0000245992.89965.94.

    Article  CAS  PubMed  Google Scholar 

  61. Denizot F, Lang R: Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 1986, 89: 271-277. 10.1016/0022-1759(86)90368-6.

    Article  CAS  PubMed  Google Scholar 

  62. Huber W, Von Heydebreck A, Sültmann H, Poustka A, Vingron M: Variance stabilization applied to microarray data calibration and to the quantification of differential expression. Bioinformatics. 2002, 18: S96-S104.

    Article  PubMed  Google Scholar 

  63. McClintick JN, Edenberg HJ: Effects of filtering by Present call on analysis of microarray experiments. BMC Bioinformatics. 2006, 7: 49-10.1186/1471-2105-7-49.

    Article  PubMed Central  PubMed  Google Scholar 

  64. Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J: TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003, 34: 374-378.

    CAS  PubMed  Google Scholar 

  65. Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA. 2001, 98: 5116-5121. 10.1073/pnas.091062498.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  66. Chu VT, Gottardo R, Raftery AE, Bumgarner RE, Yeung KY: MeV+R: using MeV as a graphical user interface for Bioconductor applications in microarray analysis. Genome Biol. 2008, 9: R118-10.1186/gb-2008-9-7-r118.

    Article  PubMed Central  PubMed  Google Scholar 

  67. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000, 25: 25-29. 10.1038/75556.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references


We thank Dr S Vilotti for kindly providing the immunofluorescence images and Dr M Biagioli for her invaluable qRT-PCR guidance. We would also like to thank Prof. G Tell for critically reading the manuscript and all members of Prof. G Tell, CT and SG laboratories for discussions. This work was supported by the Telethon Grant GGP05062, by the Regional Grant DL26 (Regione FVG, Italy) and by The Giovanni Armenise-Harvard Foundation career development award to SG.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Stefano Gustincich.

Additional information

Authors' contributions

RC performed data analysis, assisted with the generation of microarray data, validated targets by qRT-PCR, wrote and revised the manuscript. CB and PG performed the cell culture and the MTT assays. PR performed the bioinformatic and statistical analysis of the microarray experiments. RF performed some qRT-PCR experiments. HK produced the microarray data at the CBM Genomics unit (Trieste, Italy). SG conceived, designed and wrote the paper. CT and SG provided support, direction and oversight of the experiments and revised the final manuscript. All authors read and approved the final version of the manuscript.

Electronic supplementary material


Additional file 1: Table S1: Differentially expressed genes in response to 24 h of UCB treatment at Bf 140 nM in SH-SY5Y cells. Affymetrix Probe Set IDs, gene names and symbols and fold changes (FC) are indicated. (XLS 48 KB)


Additional file 2: Table S2. Identification of the GO cellular components categories of differentially expressed genes in response to UCB-treatment in SH-SY5Y cells (GO slim annotation as reported in Table 1). (XLS 30 KB)

Authors’ original submitted files for images

Below are the links to the authors’ original submitted files for images.

Authors’ original file for figure 1

Authors’ original file for figure 2

Authors’ original file for figure 3

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Calligaris, R., Bellarosa, C., Foti, R. et al. A transcriptome analysis identifies molecular effectors of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells. BMC Genomics 10, 543 (2009).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: