Impacts of ORP manipulation on SGBS cell adipogenic differentiation.

<p>ORP8 or ORP3 were overexpressed by infecting cells on day 10 with control (Blank) or ORP (Ad-ORP8, Ad-ORP3) adenoviral vectors, and collected for analysis on day 13. SGBS preadipocytes were transduced with an ORP11 shRNA (Sh-ORP11) or non-targeting (Sh-NT) shRNA lentivirus, followed by differentiation for 22 days. A. Western blots of total cell protein (10 µg/lane); ORP11, after 22 days of differentiation; ORP8 and ORP3, after 72 h of adenoviral transduction. The blots were probed with anti-β-actin as a loading control. B. The impacts of ORP11 silencing or ORP8/ORP3 overexpression on the mRNA levels of adipocyte differentiation markers adiponectin, aP2, leptin and PPARγ. In cells with ORP11 stably silenced also ORP8 and ORP3 mRNAs were quantified. The results are shown as fold changes relative to cells infected with the corresponding control viruses, and represent mean ± S.E., n = 3; *p<0.05. C. The cellular triglyceride concentration was measured by using an enzymatic assay. The results were normalized for total cell protein and are presented relative to cells infected with the corresponding control viruses (mean ± S.E., n = 3; *p<0.05, **p<0.01).</p

Time course of ORP11, ORP3, and ORP8 mRNA changes upon SGBS cell adipogenic differentiation.

<p>A. Phase contrast images of SGBS preadipocytes (0 D) and differentiating adipocytes on days 10, 14, and 22. Bars, 200 µm. B. The ORP mRNA levels were quantified at the differentiation time points indicated, as fold changes relative to preadipocytes (day 0). The data represents mean ± S.E., n = 3.</p

Adipogenic differentiation of SGBS cells.

<p>Haematoxylin and Oil Red O staining of SGBS preadipocytes (A) and adipocytes differentiated for 22 days (B). C. Relative mRNA levels of the adipocyte differentiation markers adiponectin, aP2, leptin and PPARγ in SGBS adipocytes as compared to preadipocytes. The bars indicate fold-change, mean ± S.E. (n = 3).</p

Copy numbers of OSBP/ORP mRNAs in human adipose tissues and SGBS adipocytes.

<p>The mRNAs were quantified by qPCR using the corresponding cDNAs as calibrators (see Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045352#s2" target="_blank">Methods</a>), and the mRNA quantities are presented on a log10 scale as copies/ng total RNA. A. Subcutaneous adipose tissue; B. Visceral adipose tissue. The data represents mean ± S.E., n = 4. C. Simpson-Golabi-Behmel syndrome (SGBS) adipocytes after 22-day differentiation. The mean from a single experiment carried out in triplicate is shown.</p

Changes in ORP mRNA and protein expression upon SGBS cell adipogenic differentiation.

<p>A. Quantities of the indicated ORP mRNAs on day 22 of differentiation. The results are presented as fold change relative to preadipocytes. The data represents mean ± S.E., n = 3−4, *p<0.05, **p<0.01. B. Western blot analysis of the ORP11, ORP8, and ORP3 proteins in SGBS preadipocytes and adipocytes (day 22). C. Quantification of the Western data by densitometric analysis. The ORP11, ORP8 and ORP3 signals were normalized for that of β-actin. The data represents mean ± S.E., n = 3−4, *p<0.05.</p