Transmission electron microscopy of 2D-cardiac cells.

<p>The 2D-cardiac cells are quite distinct from the 3D-cardiac cell aggregates (<b>a–b</b>), because they present several vesicles (Ve), a large endoplasmic reticulum (ER) and elongated mitochondria (M), some of which are branching, as seen in image (<b>b</b>). N, nucleus.</p

Analysis of the distribution of the cytoskeleton in 2D- and 3D-cardiac cultures.

<p>Cells were grown for 48 hours, fixed with 4% paraformaldehyde and triple-stained with Texas red-phalloidin (red), mouse monoclonal anti-α-tubulin antibody (green), and the DNA-specific probe DAPI (blue). Cells were analyzed in a Leica laser scanning confocal microscope. Different optical focal planes of the cells were acquired and projected in order to show both the 2D-cells and the 3D-aggregates (<b>a</b>). The same stack in <b>a</b> can be seen with the F-actin stain only, where 2D-cells display well organized stress fibers (arrow, <b>b</b>) whereas no detectable stress fibers are seen in the 3D-aggregates (<b>b</b>). Again, in the same stack in (<b>a</b>, <b>b</b>), microtubules and nuclei were superposed to show well spread microtubules in 2D-cells (arrow), whereas only a few cells in the surface of the aggregates display organized microtubules (<b>a, c</b>). Note MTOCs close to the nuclei in 2D-cells. The same stack can be seen in a tilted projection in (<b>d</b>), showing the relationship of 2D and 3D cells. The same stack can be seen in a lateral view, showing the volume of the aggregate and the connection between 2D and 3D-cells (<b>e</b>). Lastly, the same stack is shown in a transverse section of a lateral projection, showing the distribution of microtubules, microfilaments and nuclei inside the aggregate (<b>f</b>).</p

Transmission electron microscopy of 3D-cardiac aggregates.

<p>It is possible to notice a large number of cell-cell contacts (arrows), well-preserved mitochondria (M), and myofibrils (Mio) in a cardiac aggregate. Images (<b>b–c</b>) also show well-preserved mitochondria (M), organized myofibrils (Mio) and desmosomes (arrows), which are better visualized in image (<b>c</b>). Note in image (<b>c</b>) that some transversal sections of myofibrils (Mio) and other intercellular contacts are found (arrowheads). Image <b>d</b> shows myofibrils (Mio) with its Z lines (asterisks), as well several mitochondria (M) and a nucleus (N).</p

3D-cardiac cells form round shaped-structures while 2D-cardiac cells are flattened.

<p>3D-cardiac cell aggregates and 2D-cardiac cells were isolated from hearts of 11-day-old chick embryos by mechanical and enzymatic dissociation and plated onto collagen-coated substrates. Phase-contrast microscopy of live cells shows 3D-organized cardiac aggregates (<b>a</b>) and well spread and flattened isolated cardiac cells (<b>a–b</b>).</p

Comparison of the distribution of microtubules in 2D- and 3D-cardiac cultures.

<p>Cells were grown for 48 hours, fixed with 4% paraformaldehyde and triple-stained with polyclonal anti-connexin-43 antibody (red), mouse monoclonal anti-α-tubulin antibody (green), and the DNA-specific probe DAPI (blue). Cells were analyzed in a Leica laser scanning confocal microscope. Different optical focal planes of the cells were acquired and projected in order to show both the 2D-cells and the 3D-aggregates. 2D-cells display an extensive microtubular network (arrow, <b>a</b>), whereas only a few cells in the surface of the aggregates display organized microtubules. An intense labeling of connexin 43 is only seen in the 3D-aggregates (<b>a</b>). To highlight the differences between the 2D and 3D-cells, we projected the slices corresponding to the 3D-aggregate only in image (<b>b</b>) and the slices corresponding to the 2D-cells only in image (<b>c</b>).</p

Proliferation rate of 2D and 3D-cardiac cells.

<p>24-hs cardiac cell cultures were incubated with BrdU (3 ug/mL) and stained with anti-BrdU antibody. 2D-isolated cells were analyzed by phase contrast (<b>a–b</b>, gray) and conventional fluorescence microscopy (<b>a–b</b>, red), while 3D-aggregates were analyzed by confocal microscopy (<b>c–g</b>). Images (<b>a</b>, <b>b</b>) are different magnifications. BrdU-positive nuclei are found in both 2D and 3D-cells, but are more frequent in 2D-cells (<b>a–g</b>). Selected confocal slices, 10 µm apart, from bottom to top are shown in images (<b>c–e</b>). Note the difference in the distribution of BrdU positive cells along the aggregate Z-axis (<b>c–e</b>). All focal planes were merged in image (<b>f</b>), where it is possible to see the whole aggregate because of the faint fluorescence background. The relative position of all cells can be better visualized in the depth-color coded image shown in (<b>g</b>). The colored scale represents slice count, from bottom to top, each 2 µm apart. Scale bars in images (<b>a, b, c, f</b>) correspond to 50 µm.</p

Scanning electron microscopy of 2D-cardiac cells and 3D-cardiac aggregates.

<p>2D-cardiac cells are flattened and well-spread while a cardiac aggregate form a large cluster composed of cells in intimate contact (<b>a</b>). A large amount of extracellular matrix is seen connecting the cells in the 3-D aggregate (<b>b–c</b>). Note that the same aggregate is seen in detail after progressive higher magnifications (<b>a–c</b>). 3D-aggregates freshly isolated from chick embryo's hearts were also analyzed by SEM (<b>d–f</b>). Note the large amount of extracellular matrix found within the cardiac aggregates (<b>e–f</b>).</p

Distribution of flotillin-2 in human neonatal muscle cells.

<p>Human neonatal muscle cells were grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103990#s2" target="_blank">Materials and Methods</a> section. Cells were fixed with methanol and stained with an anti-flotillin-2 antibody (<b>green</b>, <b>A</b> and <b>B</b>) and the nuclear dye DAPI (<b>blue</b>, <b>A</b> and <b>B</b>). Merged images are shown in <b>A</b> and <b>B</b>. Note that flotillin-2 is present in human myoblasts and myotubes in vesicle-like structures (arrows in <b>A</b> and <b>B</b>). Scale bar in <b>A</b> represents 10 µm.</p

Flotillin-2 is down-regulated during <i>in vitro</i> chick skeletal myogenesis.

<p>Chick myogenic cells were grown for 24, 48 and 72-2. (<b>A</b>) Upper Western blot shows flotillin-2 reactivity and lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading. (<b>B</b>) Quantification of protein bands revealed a progressive decrease in the levels of flotillin-2 expression during skeletal muscle differentiation. *p<0.05; ANOVA followed by Tukey post hoc test versus 24-h group, n = 3.</p

Cholesterol depletion enhances the expression of flotillin-2 protein and mRNA.

<p>Chick myogenic cells were grown 24(control, <b>Ct</b>). Cell culture extracts were analyzed in Western blot using an antibody against flotillin-2 (<b>A</b>). Lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading (<b>A</b>). Quantification of protein bands revealed a 40% increase in the levels of flotillin-2 expression after cholesterol depletion (<b>B</b>). RT-PCR analysis (for details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103990#s2" target="_blank">Materials and Methods</a>) of the expression of flotillin-2 in control and in MbCD-treated cells is shown in <b>C</b>. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. Analysis of the expression of flotillin-2 shows a more than 2-fold increase in the levels of mRNA expression in MbCD-treated cells compared with control cells. *p<0.05; t test for unpaired samples, n = 3.</p