Clinical features present in carriers of <i>JAG1</i> mutations at the time of hospitalization for neonatal cholestasis and at 3 years of age.

<p>* siblings;</p><p>ERCP, endoscopic retrograde cholangiopancreatography; BA, biliary atresia; BA type 3 –gallbladder, cystic duct and common bile duct are patent; BA type 4 –atresia of all the extrahepatic bile ducts; clinical features were present (+) or missing (-),</p><p>^# indicates clinical features not present at the age of 2 months;</p><p>AGS criteria indicate the number of major clinical features (diagnostic criteria) of Alagille syndrome present at the age of 2 months and 3 years, respectively.</p><p>Clinical features present in carriers of <i>JAG1</i> mutations at the time of hospitalization for neonatal cholestasis and at 3 years of age.</p

Putative transcription factor binding sites and conservation of <i>ATP8B1</i> 5′UTR.

<p>(A) Sequence alignment using ClustalW algorithm (<a href="http://www.ebi.ac.uk/Tools/clustalw/" target="_blank">http://www.ebi.ac.uk/Tools/clustalw/</a>). High level of conservation among mouse (M), rat (R) and human (H) genome was detected for Ex −3 and Ex −4 (conserved nucleotides indicated by stars). Putative Sp-1, Ap-2, NFκB transcription factor binding sites were predicted in exonic/promoter region P3. Putative CREB and HNF-4 binding sites were identified within a distal part of promoter P3 corresponding to Ex −4 sequence. Initiator element sequence encompasses the main TSS cluster in Ex −3. Exonic regions are underlined, transcription start sites are indicated by arrows and bold letters and putative transcription factor binding sites by grey boxes. Two upstream ATG are in bold. (B) Sequence of Ex −2 and (C) Ex −1. Exonic region is highlighted in bold. Alu consensus sequences are underlined.</p

Schematic representation of the Jagged1 protein and spliced <i>JAG1</i> mRNA with mutations found in our patients.

<p>Schematic representation of the Jagged1 protein and spliced <i>JAG1</i> mRNA with mutations found in our patients.</p

Functional analysis of <i>ATP8B1</i> promoter regions.

<p>HepG2 cells were transiently transfected with luciferase reporter gene constructs containing 12 different fragments of putative <i>ATP8B1</i> promoters. Nine luciferase constructs (Prom 1 to Prom 9) were designed to comprise the putative dominant promoter P3, two constructs covered promoter P1 (Proms 11 and 12) and one covered promoter P2 (Prom 10). The position of the tested fragments are indicated by horizontal double arrow lines. The number in brackets next to the construct name represents its size (bp). Prom 3 and Prom 4 were designed to include/exclude a putative FXR/RXR binding site indicated by black oval. Antisense construct encodes the same region as Prom 5, but in antisense orientation. Putative promoters (P1–P3) are depicted as horizontal thick arrows. Transcriptional activity for each construct was measured in relative light units per second (RLU/s) and corrected for the transfection efficiency using the internal control <i>Renilla</i> pRL-TK expression plasmid. The data shown are calculated from 3–5 independent experiments and related to the pGL3 Basic activity.</p

Thermodynamic properties of identified 5′UTR isoforms.

<p>The comparison of the 5′UTR length and RNA secondary structure free energy and percentage of minimal free energy (MFE) for all identified <i>ATP8B1</i> 5′UTR isoforms, schematically depicted on the left. Prediction for the most frequent isoforms initiating at Ex −3 was calculated using TSS at position −125 from 3′end of Ex −3. Data in brackets (row 3 and 4) represent data for TSS at position −509. Putative secondary RNA structures predicted using RNAfold web tool (<a href="http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi" target="_blank">http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi</a>) are summarised in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051650#pone.0051650.s002" target="_blank">Fig. S2</a>.</p

The heterogeneity of 5′UTR of <i>ATP8B1</i> gene.

<p>(A) Length and position of novel untranslated exons of <i>ATP8B1</i> gene. (B) Six identified alternatively spliced variants are indicated by diagonal lines. (C) The existence of two acceptor splice sites CAGCAG (tandem acceptors) at the 5′ boundary of the first translated exon (Ex +1) of <i>ATP8B1</i> allows the generation of two different splice forms for each combination of upstream exons with Ex +1. Thus generated splice forms differ from each other by only three nucleotides CAG and give rise to twelve <i>ATP8B1</i> isoforms in total (D).</p

Mutations in <i>JAG1</i> found in patients with biliary atresia and Alagille syndrome.

<p>* siblings;</p><p>AGS, Alagille syndrome; BA, biliary atresia; novel mutations are in <b>bold</b>.</p><p>Mutations in <i>JAG1</i> found in patients with biliary atresia and Alagille syndrome.</p

<i>ATP8B1</i> promoter activity in cells stimulated with bile acids.

<p>No significant change in activity was detected after stimulation with CDCA. (A) HepG2 cells were transiently transfected with three previously characterised (Fig. 4) <i>ATP8B1</i> promoter gene constructs (Prom 3, 4 and 6) and stimulated with 0, 10, 50 and 100 µM CDCA for 24 hours. All constructs comprise proximal 434 bp-promoter P3, Prom 4 includes putative FXR binding site identified by MatInspector computer analysis software, and Prom 6 represents the largest construct containing 3379 bp of 5′flanking region. (B) HepG2 cells stably expressing rat sodium-taurocholate co-transporting polypeptide (rNtcp) were transiently transfected with constructs Prom 3, 4 and 6 together with 50 ng of pCI_hRXRα and 50 ng pCI_hFXR plasmids and treated with 0, 10 and 25 µM CDCA for 24 hours.</p

Relative expression levels of different splicing forms assessed by qRT-PCR.

<p>Diagram of individually designed probes (for probe and primer sequences see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051650#pone.0051650.s003" target="_blank">Table S1</a>) used to evaluate the expression levels of twelve identified splicing variants of <i>ATP8B1</i> 5′UTR. Tested splicing variants are indicated by Latin numbers on the left, average expression levels for each transcript from normal liver tissues (n = 7) are shown on the right. The expression levels are presented as a relative value normalised to the expression of the protein coding region represented by Ex +1/Ex +2 boundary.</p

Mechanical Regulation of Mitochondrial Dynamics and Function in a 3D-Engineered Liver Tumor Microenvironment

It has become evident that physical stimuli of the cellular microenvironment transmit mechanical cues regulating key cellular functions, such as proliferation, migration, and malignant transformation. Accumulating evidence suggests that tumor cells face variable mechanical stimuli that may induce metabolic rewiring of tumor cells. However, the knowledge of how tumor cells adapt metabolism to external mechanical cues is still limited. We therefore designed soft 3D collagen scaffolds mimicking a pathological mechanical environment to decipher how liver tumor cells would adapt their metabolic activity to physical stimuli of the cellular microenvironment. Here, we report that the soft 3D microenvironment upregulates the glycolysis of HepG2 and Alexander cells. Both cell lines adapt their mitochondrial activity and function under growth in the soft 3D microenvironment. Cells grown in the soft 3D microenvironment exhibit marked mitochondrial depolarization, downregulation of mitochondrially encoded cytochrome c oxidase I, and slow proliferation rate in comparison with stiff monolayer cultures. Our data reveal the coupling of liver tumor glycolysis to mechanical cues. It is proposed here that soft 3D collagen scaffolds can serve as a useful model for future studies of mechanically regulated cellular functions of various liver (potentially other tissues as well) tumor cells