Flotillin-2 is found in pre-fusion myoblasts after cholesterol depletion.

<p>Immunofluorescence of flotillin-2, desmin and DAPI in cultures treated with methyl-β-cyclodextrin (MbCD) showing flotillin-2 expression in mononucleated cells that are fusing with myotubes. Chick myogenic cells were grown for 24 hours, treated with 2 mM MbCD for 30 min and grown for the next 24 hours (<b>B</b> and <b>C</b>). Some cells were not treated and were fixed at 48 hours of culture (control, <b>A</b>). All cells were fixed with paraformaldehyde and stained with antibodies against desmin (<b>red</b>) and flotillin-2 (<b>green</b>) and with the nuclear dye DAPI (<b>blue</b>). Merged images are shown in <b>A–C</b>. Note an increase in the number of flotillin-2 positive-mononucleated cells in close contact with the membrane of multinucleated myotubes (arrows in <b>B</b> and <b>C</b>). Scale bar in <b>A</b> represents 20 µm.</p

Flotillin-2 distribution in multinucleated myotubes.

<p>Myogenic cells were grown for 72-flotillin-2 antibodies (green, <b>A</b> and <b>C</b>) and the nuclear dye DAPI (blue, <b>B</b> and <b>D</b>). Note that with methanol fixation it is possible to see that flotillin-2 is present in elongated dots in myotubes (arrows in <b>A</b> and <b>C</b>). Scale bar in <b>D</b> represents 20 µm.</p

Flotillin-2 distribution in myogenic cells during skeletal muscle differentiation.

<p>Myogenic cells were grown for 24(<b>A</b> and <b>B</b>) or 72 hours (<b>C</b>). Cells were fixed with paraformaldehyde and stained with antibodies against desmin (<b>red</b>, <b>A</b> and <b>C</b>) and flotillin-2 (<b>green</b>, <b>A, B</b> and <b>C</b>) and with the nuclear dye DAPI (<b>blue</b>, <b>A</b> and <b>C</b>). Merged images are shown in <b>A</b> and <b>C</b>. Note that with paraformaldehyde fixation it is possible to see flotillin-2 almost exclusively in mononucleated cells in vesicle-like structures (<b>B</b>) and nearly absent from myotubes (<b>A</b> and <b>C</b>). Scale bar in <b>A</b> and <b>C</b> represents 20 µm and in <b>B</b> represents 10 µm.</p

Brefeldin A induces a major reduction in the number of flotillin-2 containing vesicles.

<p>Chick myogenic cells were grown for 24(5 µg/ml) for 3 hours or with nocodazole (10 µg/ml) for 3 hours. After treatment, cells were grown in fresh culture medium for the next 3 hours. In images (<b>A–F</b>) cells were fixed with paraformaldehyde and stained with an antibody against flotillin-2 (<b>green, A,C,E</b>) and with the nuclear dye DAPI (<b>blue, B,D,F</b>). Note that while nocodazole have no effect in the presence and distribution of flotillin-2 positive vesicles (<b>C,D</b>), brefeldin A induced a major reduction in flotillin-2 containing vesicles (<b>E,F</b>). In images (<b>G–I</b>) cells were analyzed under phase contrast microscopy and superimposed with DAPI (blue). Scale bars represents 50 µm (in <b>A–F</b> and <b>G–I</b>).</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

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

Transmission electron microscopy of chick fibroblastic cells.

<p>Chick myogenic cells were grown for 48(asterisks), well-preserved mitochondria (M), as well as several endoplasmic reticulum membranous profiles (ER) and a nucleus (N). Image shown in <b>b</b> is a higher magnification of a region of the image shown in <b>a</b>. Scale bar in <b>a</b> represents 1 µm and in <b>b</b> represents 500 <i>µ</i>m.</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

Cryo-immunogold EM labeling of flotillin-2 in myogenic cells.

<p>Chick myogenic cells were grown for 48-flotillin-2 antibody followed by a 10-<i>η</i>m gold-conjugated secondary antibody. Note that gold particles are present in vesicles (<b>a</b>) and associated with Golgi stacks (<b>b</b>). GC, Golgi complex. Scale bar in <b>a</b> represents 100 <i>η</i>m and in <b>b</b> represents 250 <i>η</i>m.</p

microRNAs exclusively expressed in FSHD biopsies.

<p>microRNAs exclusively expressed in FSHD biopsies.</p