LXRα Enhances Lipid Synthesis in SZ95 Sebocytes Il Hong, Min-Ho Lee, Tae-Young Na, Christos C. Zouboulis, Mi-Ock Lee  Journal of Investigative Dermatology  Volume 128, Issue 5, Pages 1266-1272 (May 2008) DOI: 10.1038/sj.jid.5701134 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 TO901317 induces lipid synthesis in the SZ95 sebocytes. (a) The SZ95 sebocytes were treated with vehicle (upper left panel), 100μM linoleic acid (upper right panel), or 1μM TO901317 (lower left panel) for 48hours. Also, 1μM 9-cis-RA was co-treated with 1μM TO901317 (lower right panel). At the end of treatment, lipid droplets in the SZ95 sebocytes were stained by Oil Red O and examined by a light microscope. Bar=50μm. (b) The SZ95 sebocytes were treated with vehicle (left panel) or 1μM TO901317 (right panel) for 48hours. Lipid droplets in cells were stained by Nile red and examined by a fluorescence microscope using a 530–550nm bandpass exciter filter by light emission of >575nm. Bar=50μm. (c) The SZ95 sebocytes were treated with vehicle (green), 10μM 22(R)-HC (purple), 1μM TO901317 (blue), or 100μM linoleic acid (black). The histogram obtained with sebocytes without staining was filled with red. Flow cytometry was performed after cells were stained with Nile red. Fluorescence was measured by a 580±30nm band pass filter. One representative of at least three independent experiments with similar results is shown. Journal of Investigative Dermatology 2008 128, 1266-1272DOI: (10.1038/sj.jid.5701134) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 TO901317 induces the expression of LXRα and its downstream target genes in the SZ95 sebocytes. The SZ95 sebocytes were treated with 1μM TO901317 for the indicated time periods. (a) The expression of LXRα, LXRβ, and SREBP-1 (65kDa) proteins was analyzed by western blot analysis using specific antibodies. The expression of α-tubulin was monitored as a control. One representative of at least three independent experiments with similar results is shown (upper panel). The density of the indicated protein band was determined using an image analysis system. The value was normalized to that of α-tubulin and expressed as fold induction relative to vehicle treatment. Data shown are the mean±SD of three independent experiments (lower panel) (*P<0.05; **P<0.01). (b) The expression of LXRα, LXRβ, SREBP-1c, and FAS transcripts was analyzed by real-time PCR. Data shown are the mean±SD of three independent experiments (*P<0.05; **P<0.01; ***P<0.001). Journal of Investigative Dermatology 2008 128, 1266-1272DOI: (10.1038/sj.jid.5701134) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 LXRα mediates the response of the SZ95 sebocytes to TO901317. (a) The SZ95 sebocytes were transfected with 1μg LXRE-Luc in the absence or presence of 20ng pCMX-LXRα or pCMX-LXRβ. After 24hours of transfection, cells were treated with vehicle or with the indicated concentration of TO901317 for 24hours. At the end of treatment, cell lysates were obtained and analyzed. Data shown are the mean±SD of three independent determinations (*P<0.05; **P<0.01; ***P<0.001). (b) The SZ95 sebocytes were transfected with 3 μg each p3XFLAG7.1-AS-LXRα (AS-LXRα) or empty vector (EV). After 24hours of transfection, cells were treated with or without 1μM TO901317 for 24hours. At the end of treatment, the expression of LXRα, LXRβ, and SREBP-1 was analyzed by western blot analysis. One representative of three independent experiments with similar results is shown (left panel). The density of the indicated protein band was determined using an image analysis system. The value was normalized to that of α-tubulin and expressed as fold induction relative to no treatment. Data shown are the mean±SD of three independent experiments (right panel) (*P<0.05; ***P<0.001). (c) The SZ95 sebocytes were transfected with 2μg each empty vector (upper panel) or p3XFLAG7.1-AS-LXRα (lower panel). After 24hours of transfection, cells were treated with (right panel) or without (left panel) 1μM TO901317 for 48hours. At the end of treatment, lipid droplets in the SZ95 sebocytes were examined by a light microscope. Bar=50μm. One representative of at least three independent experiments with similar results is shown. Journal of Investigative Dermatology 2008 128, 1266-1272DOI: (10.1038/sj.jid.5701134) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 TO901317 induces the expression of PPAR subtypes in the SZ95 sebocytes. The SZ95 sebocytes were treated with 1μM TO901317 for the indicated time periods. (a) The expression of PPARα, PPARβ, and PPARγ proteins was analyzed by western blot analysis using specific antibodies. The expression of α-tubulin was monitored as a control. One representative of at least three independent experiments with similar results is shown (upper panel). The density of the indicated protein band was determined using an image analysis system. The value was normalized to that of α-tubulin and expressed as fold induction relative to vehicle treatment. Data shown are the mean±SD of three independent experiments (lower panel) (*P<0.05; **P<0.01; ***P<0.001). (b) The transcripts of PPARδ and PPARγ and their down stream genes, phosphoenolpyruvate carboxykinase-2 (PEPCK-2) and acyl Coenzyme A synthetase-2 (ACS-2), were analyzed by real-time PCR. Data shown are the mean±SD of three independent experiments (*P<0.05; **P<0.01; ***P<0.001). Journal of Investigative Dermatology 2008 128, 1266-1272DOI: (10.1038/sj.jid.5701134) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 TO901317 represses the expression of COX-2 and iNOS. The SZ95 sebocytes were treated with 10ng/ml LPS in the presence or absence of 1μM TO901317 for 24hours. The expressions of COX-2 and iNOS protein were analyzed by western bolt analysis using specific antibodies. The expression of α-tubulin was monitored as a control. One representative of two or three independent experiments with similar results is shown (upper panel). The density of the indicated protein band was determined using an image analysis system. The value was normalized to that of α-tubulin and expressed as fold induction relative to vehicle treatment. Data shown are the mean±SD of two or three independent experiments (lower panel) (*P<0.05). Journal of Investigative Dermatology 2008 128, 1266-1272DOI: (10.1038/sj.jid.5701134) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions