Cavin-2 is degraded by the proteosome and Cavin-1 redistributes to the cytosol following cholesterol depletion.

<p>3T3-L1 adipocytes were treated with or without MβCD (20 mM, 90 minutes) and subjected to either (<b>A</b>) immunostaining with the indicated antibodies (red) and for nuclei with DAPI (4′,6-diamidino-2-phenylindole, blue) and analyzed by confocal microscopy as described in Methods or (<b>B</b>), subjected to subcellular separation into membrane and cytosolic fractions. Following centrifugation, an equal proportion of each fraction (ca. 6X more cytosol protein than membrane) were analyzed by SDS-PAGE and Western blotting. Glyceraldehyde phosphate dehydrogenase (GAPDH) is a loading control for the cytosolic fraction. (<b>C</b>) Cultured at cells were treated with or without methylβ-cyclodextrin in combination with the proteasome inhibitor MG-132 (10 µM), the lysosomal inhibitor chloroquine (40 µM), or the inhibitors alone for 90 minutes and cell extracts were prepared in lysis buffer and analyzed by SDS-PAGE and Western blotting for the proteins indicated. Shown are representative experiments.</p

Cholesterol depletion by simvistatin and cholesterol-binding antibiotics causes loss of cavin-2 in fat cells (A) and fibroblasts (B).

<p>Adipocytes or NIH-3T3 fibroblasts were treated with or without Simvastatin (10 µM, 18 hrs) methyl-β-cyclodextrin (20 mM, 90 minutes), nystatin (76 µM, 4 hours) or filipin (7.6 µM, 4 hours). Whole cell extracts were then prepared in RIPA buffer and analyzed by SDS-PAGE and Western blotting as in prior figures.</p

Cholesterol repletion restores cavin-2 levels, which allows return of cavin-1 to the plasma membrane.

<p>3T3-L1 adipocytes were treated without or without methyl-β-cyclodextrin (20 mM, 90 minutes), and the medium was changed to that containing either 10% FBS, 10% FBS depleted of lipoproteins (LPDS), or, 10% FBS lipoprotein depleted serum containing cholesterol loaded cyclodextrin (25 µM cholesterol). Following incubation for the times indicated (time 0 being after MβCD removal), cell lysates were analyzed by SDS-PAGE and Western blotting (<b>A</b>) or were processed for immunofluorescence with antibodies for cavin-1 and -2 (<b>B</b>) and for nuclei (DAPI) as in prior figures. The percent cavin-2 rim staining was determined by scoring 104+/−3 cells that were also positive for DAPI staining.</p

ShRNA mediated knockdown of cavin-2 causes redistribution of cavin-1 to the cytosol.

<p>3T3-L1 adipocytes stably expressing shRNA directed against enhanced green fluorescent protein (eGFP) or cavin-2 were processed for freeze drying electron microscopy (<b>A</b>) or subjected to immunostaining (<b>B</b>) with the indicated antibodies (red) and DAPI (blue), and analyzed by confocal microscopy and quantitatively analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034516#pone-0034516-g003" target="_blank">Figure 3</a> for 102 cells or (<b>C</b>) subcellular fractionation into membrane and cytosolic fractions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034516#pone-0034516-g002" target="_blank">Figure 2B</a>. Following fractionation, proteins were analyzed by SDS-PAGE and Western blotting, with glyceraldehyde phosphate dehydrogenase (GAPDH) being used as a loading control for the cytosolic fraction. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034516#pone-0034516-g004" target="_blank">Figure 4A</a>, panel B, black arrows point to torus shaped caveolae and white arrows to flattened caveolae and the scale bar is 200 nm. In 4B, the amount of cavin-2 in whole cell lysates is shown. These are representative of 3 such experiments.</p

Cav1 null adipocytes lack caveolae but cavin-2 is membrane associated and redistributes to the cytosol upon cholesterol depletion.

<p>Cav1 null adipocytes were treated with or without MβCD as in previous figures and lysates (<b>A</b>) were prepared and analyzed by Western blot or (<b>B</b>) labeled with anti-cavin-2 and secondary antibody prior to analysis by confocal microscopy.</p