Supplemental Material

This article contains the following supporting material:

• Figure 1 - The decellularization protocol effectively removes cells and cellular debris but not ECM components. Cells were cultured on glass coverslips, subjected to a decellularization procedure with distilled water as described in Materials and Methods (D-F), or not (A-C) . The coverslips with or without cells were subsequently incubated with 4% PFA at room temperature for 20 min followed by labeling with the nuclear stain DAPI (B,E) and anti-fibronectin antibodies (see Materials and Methods; C,F). A and D are phase contrast images to B,C and E,F, respectively. Note that the decellularization protocol effectively removes cells (D versus A) and DNA residues (E versus B) but not the ECM protein fibronectin (F versus C). In an other experiment, cells were cultured on glass coverslips, subjected to a decellularization procedure with destilled water as described in Materials and Methods (H-J) , or not (G) , after which the coverslip was processed for scanning electron microscopy and analyzed. Note the absence of cells and cellular debris after decellularization (H versus G), and the detection of fibrillar ECM-like structures (I,J) .

• Figure 2 - The multicellular apical lumen is enclosed within the cells. Cells cultured on predeposited ECM for 72 h were fixed and (immuno)labeled with antibodies against the canalicular surface marker MRP2 (A) and TRITC-labeled phalloidin (B) to stain the actin cytoskeleton. Cells were then analyzed by laser scanning confocal microscopy. Serial x-y sections (1 per 0.5 μm) were taken and x-z images were reconstructed of the coordinates indicated by the dashed crosshair in the x-y panels. Note that the apical lumen is enclosed within the cell cluster. Bar 10μm. Animated scrolling through the serial x-y sections and a 3D animated projection is shown in supplementary movies M1 and M2, respectively.

• Figure 3 - Blebbistatin stimulates cell clustering and apical lumen remodeling. Cells were cultured on glass coverslips in the presence of 100 μmM of the specific myosin-II inhibitor blebbistatin for 72 h. Cells were then fixed with 4% PFA and (immuno)labeled with anti-MRP antibodies (B) and the nuclear stain DAPI (A), and subjected to laser scanning confocal microscopical. Multiple x-y sections (1 per 0.5 μm) were superimposed. A merged image is shown in C. Note the extensive clustering of the cells and the appearance of a large multicellular lumen within the cell cluster. Bar 10 μm.

• Movie 1 - Animated scrolling through serial x-y sections of a cell cluster with a multicellular lumen. Cells cultured on predeposited ECM for 72 h were fixed and (immuno)labeled with antibodies against the canalicular surface marker MRP2 and TRITC-labeled phalloidin to stain the actin cytoskeleton. Cells were then analyzed by laser scanning confocal microscopy. Serial x-y sections (1 per 0.5 μm) were taken and a movie clip showing the serial x-y sections from top to bottom was prepared using Leica confocal software Lite v2.61. An animated 3D projection of the serial x-y sections is shown in supplementary movie M2.

• Movie 2 - Animated 3D projection of a cell cluster with a multicellular lumen. Cells cultured on predeposited ECM for 72 h were fixed and (immuno)labeled with antibodies against the canalicular surface marker MRP2 and TRITC-labeled phalloidin to stain the actin cytoskeleton. Cells were then analyzed by laser scanning confocal microscopy. Serial x-y sections (1 per 0.5 μm) were taken and an animated 3D projection was prepared using Leica confocal software Lite v2.61. Animated scrolling through the serial x-y sections that were the basis of this 3D projection is shown in supplementary movie M1.

• Movie 3 - Animated scrolling through serial x-y sections of a cell cluster with a multicellular lumen. Cells cultured on glass coverslips for 72 h were fixed and (immuno)labeled with antibodies against the canalicular surface marker MRP2 and TRITC-labeled phalloidin to stain the actin cytoskeleton. Cells were then analyzed by laser scanning confocal microscopy. Serial x-y sections (1 per 0.5 μm) were taken and a movie clip showing the serial x-y sections from top to bottom was prepared using Leica confocal software Lite v2.61.