Spheroids’ mounting was carried out according to the protocol described by Weiswald et add to favorites al [59]. For 3D reconstruction, a stack of confocal images was collected through the spheroids with step size of 0.488 ��m between adjacent optical planes, starting from one pole of the spheroids. 360�� 3D projects plugging from ImageJ was used to generate a 3D animation. Two-dimensional difference gel electrophoresis (2D-DIGE) analysis After acetone precipitation, protein samples (Control cells at 0 and 21 days of culture and hepatocytes obtained by CM1 or CM2) were solubilized in 2-D DIGE sample buffer: 7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, buffered to pH 8. Protein concentration was determined using the Bradford’s assay (Bio-Rad).

Then, 50 ��g protein was labelled with 400 pmol of CyDye DIGE Fluor minimal dyes (GE Healthcare) and incubated on ice in the dark for 30 min according to the manufacturer’s instructions (Cy3, Cy5 for samples and Cy2 for internal control consisting of a mixture composed by equal amounts of protein from all samples). Paired samples were reverse-labeled in order to prevent potential dye labeling bias. The reaction was stopped by addition of 1 ��l of 10 mM lysine and incubated on ice for 10 min. Samples were cup-loaded onto IPG strips, 24 cm, pH 3�C11NL (GE Healthcare), and subjected to isoelectrofocusing (IEF) in IPGphor? IEF System (GE Healthcare) according to the manufacturer’s recommendations. Upon IEF, strips were incubated in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, a trace of bromophenol blue), containing 0.

5% DTT for 15 min and thereafter in the same buffer with 4.5% iodoacetamide for 15 min. For the second dimension, strips were loaded on top of 12.5% polyacrylamide gels and run (1 W/gel) for 12�C14 h until the bromophenol blue dye reached the gel bottom-end. Subsequently, 2D gels were scanned using a TyphoonTM Trio Imager (GE Healthcare) at 100 ��m resolution with ��ex/��em of 488/520, 532/580, and 633/670 nm for Cy2, Cy3, and Cy5 respectively. The photomultiplier tube was set to ensure that the maximum pixel intensity was between 90,000 and 99,000 pixels. Image analysis was performed using DeCyder 6.5 software (GE Healthcare) as described in the user’s manual. Three independent experiments were performed for each experimental setup.

Briefly, the differential in-gel analysis module was used for spot detection, spot volume quantification Anacetrapib and volume ratio normalization of different samples in the same gel. Then the Biological Variation Analysis (BVA) module was used to match protein spots among different gels and to identify protein spots that exhibit significant differences. Manual editing was performed in the BVA module to ensure that spots were correctly matched between different gels, and to get rid of streaks and speckles. Differential expressed spots were considered for MS analysis when the fold change was larger than 1.