biof1794-sup-0001-supinfo.docxWord 2007 document , 52.9 KB

Appendix S1. Supporting Information

biof1794-sup-0002-FigureS1.tiffTIFF image, 15 MB

Figure S1 (A) Food intake and (B) body weights of C57BL/6 male mice fed a pair-fed liquid control diet (pair-fed, n=6), pair-fed + AC261066 (AC261)(10mg/kg/day, pair-fed + AC261, n=4), or a 5% v/v ethanol liquid diet (5% EtOH) and treated with either vehicle (0.1% DMSO) (5% EtOH, n = 10), or AC261 (10 mg/kg/bw/day, 5% EtOH + AC261, n=10), during liquid diet and EtOH adaptation phases, and for 3 weeks of treatments. (C) Blood alcohol content (BAC) in mice 2 h after peak blood alcohol content relative to pair-fed mice described in (A). All data errors bars represent ±SD, with ND = not detected.

biof1794-sup-0003-FigureS2.jpgJPEG image, 285.8 KB

Figure S2 Enzyme classes and proteins involved in the canonical retinoid metabolism and signaling pathway. Source: Clugston and Blaner.7

biof1794-sup-0004-FigureS3.jpgJPEG image, 488.4 KB

Figure S3 Changes in hepatic mRNA levels of subset of the 68 canonical-retinoid genes, compared to pair-fed, in pair-fed + AC261 (blue bars), 5% EtOH-treated (dark green bars), and 5% EtOH + AC261 (orange bars). Gene expression histograms are mean DEG animal values expressed as differentially expressed genes (DEG), with means expressed as log2 fold change ±SD, and # = p adjusted <0.05 compared to pair-fed mice, and * = p adjusted <0.05 in 5% EtOH + AC261 compared to 5% EtOH-treated mice.

biof1794-sup-0005-FigureS4.jpgJPEG image, 322 KB

Figure S4 (A–H) Changes in hepatic mRNA levels of retinyl ester hydrolyses (REH), compared to pair-fed (light green bars), in pair-fed + AC261 (blue bars), 5% EtOH-treated (dark green bars), and 5% EtOH + AC261 (orange bars). Gene expression histograms are mean DEG animal values expressed as differentially expressed genes (DEG), with means expressed as log2 fold change ±SD, and # = p adjusted <0.05 compared to pair-fed mice and * = p adjusted <0.05 in 5% EtOH + AC261 compared to 5% EtOH-treated mice. (I) Western blot gel and (J) western blot semi-quantitative histograms of hepatic expression of CES1. Histogram data points represent individual animal values of gel band densitometry expressed as the ratio of CES1/β-actin. All errors bars expressed as ±SD, with *p < 0.05.

biof1794-sup-0006-FigureS5.jpgJPEG image, 194.4 KB

Figure S5 AC261 treatment does not preserve hepatic retinoid levels. Quantitation of liver retinol, retinyl-palmitate, retinyl-oleate, by liquid chromatography–tandem mass spectrometry (LC–MS/MS), and liver AC261066 (AC261) and serum retinol by high performance liquid chromatography (HPLC). All errors bars expressed as ±SD, with *p < 0.05, **p < 0.01. ***p < 0.001, or ns = not significant.

biof1794-sup-0007-FigureS6.jpgJPEG image, 259.5 KB

Figure S6 (A, B, D–G) Changes in hepatic mRNA levels of retinoic acid receptors α, β, γ (RARα, β, γ), and retinoid x receptors α, β, γ (RXR α, β, γ), compared to pair-fed, in pair-fed + AC261 (blue bars), 5% EtOH-treated (dark green bars), and 5% EtOH + AC261 (orange bars). Gene expression histogram data points represent individual animal values expressed as differentially expressed genes (DEG) with means expressed as ±SD, and # = p adjusted <0.05 compared to pair-fed mice, ns = not significant, and * = p adjusted <0.05 in 5% EtOH + AC261 compared to 5% EtOH-treated mice. (C) qPCR analysis of hepatic mRNA levels of RARβ2 expressed as relative mRNA fold change compared to pair-fed mice. qPCR means expressed as ±SD with *p < 0.05 and **p < 0.01.