Volcano plots of whole body, plasma and hepatic features in the blockade arm, including the RD, WDD, WDB and WDO Groups.

<p>The analysis examines the overall impact of the DHA-mediated blockade of NASH; and includes the WDD versus RD <b>[A],</b> WDD versus WDB <b>[B]</b> and WDD versus WDO <b>[C].</b> As above, the volcano plots were created using the statistical package in MetaboAnalyst 3.0 (<a href="http://www.metaboanalyst.ca/MetaboAnalyst/" target="_blank">http://www.metaboanalyst.ca/MetaboAnalyst/</a>) as well as MS-Excel and used the data represented in the heat map (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173376#pone.0173376.g004" target="_blank">Fig 4</a></b>). The Pie Plots <b>[D]</b> represent a summary of the features that increased, decreased or did not change with diet treatment in each comparison.</p

Analysis of hepatic fatty acids: Remission arm.

<p>Hepatic fatty acids were quantified as described in Materials and Methods. <b>A:</b> The sum of fatty acids in the 4 lipid classes (saturated (SFA), monounsaturated (MUFA), ω3 and ω6 polyunsaturated fatty acids (PUFA) is presented in a stacked histogram. <b>B-D</b>: Specific fatty acids in each class are quantified and presented as μmol/mg protein. E: The mole ratio of the sum of ω3 and ω6 PUFA. Mean ± SD with 4–7. *, p<0.05 versus the RD group; #, p<0.05 versus the WDO group using one-way ANOVA.</p

Volcano plots of whole body, plasma and hepatic features in the remission arm, including the RD, WDChO and WDChD groups.

<p>As described above, volcano plots were used to describe the capacity of chow diet with and without DHA to return whole body, plasma and hepatic features to levels seen in the RD group. The comparisons included: WDChO versus RD <b>[A]</b> and WDChD versus RD <b>[B]. [C]</b> The pie plots represent a summary of the features that increased, decreased or did not change with diet treatment.</p

Diet effects on hepatic morphology: Remission arm.

<p>Livers sections from the 5 treatment groups (RD, WDB, WDO, WDChO and WDChD) in the Remission Arm were stained with trichrome and photographed at 4x. Lipid droplets appear as white circles, while branching fibrosis appear as blue strands (yellow arrows) in the trichrome stained liver sections. The slides are representative of multiple sections of each liver and all livers in each group. PT, portal track; CV, central vein.</p

Study design to examine the reversibility of NASH in male <i>Ldlr</i> ^<i>-/-</i> mice.

<p>The study design included two treatment arms; a blockade arm and a remission arm. Mice were initially randomized to 2 groups: <b>a</b>) mice maintained on chow for 30 weeks, reference diet (<b>RD,</b> n = 5); <b>b</b>) mice maintained on the western diet (<b>WD</b>) for 22 wks. After 22 wks on the WD, four mice were euthanized for blood and liver collection; these mice served as the baseline group (<b>WDB,</b> n = 4). The remaining WD-fed mice were randomized to 4 groups: <b>1</b>) mice fed the western diet + olive oil for 8 wks (<b>WDO,</b> n = 6), <b>2</b>) mice fed the WD + DHA for 8 wks (<b>WDD,</b> n = 6); <b>3</b>) mice fed the chow diet + olive oil for 8 wks (<b>WDChO,</b> n = 6); <b>4</b>) mice fed the chow diet + DHA for 8 wks (<b>WDChD,</b> n = 7). At termination of the study, mice in the RD, WDO, WDD, WDChO and WDChD groups were euthanized for liver and blood collection.</p

Overview of diet effects on anthropometric, plasma, hepatic gene expression and lipid parameters in the remission arm.

<p>As described in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173376#pone.0173376.g004" target="_blank">Fig 4</a></b>, all anthropometric, plasma, gene expression and hepatic lipid data was assembled into an excel spread sheet for each mouse in each of the 4 groups. The data was analyzed using (<a href="http://www.metaboanalyst.ca/MetaboAnalyst/" target="_blank">http://www.metaboanalyst.ca/MetaboAnalyst/</a>) to create a heat map <b>[A]</b> and carry out a principal component analysis <b>[B]</b> and hierarchical clustering using Spearman’s ranked correlation <b>[C].</b> The heat map is a visualization of the changes in abundance/level of features for each animal identification numbers are listed on the right side of the heat map. The color ranges from deep orange (high abundance or level) to deep blue (low abundance or level); white represents no change.</p

Overview of diet effects on anthropometric, plasma, hepatic gene expression and lipid parameters in the blockade arm.

<p>All anthropometric, plasma, gene expression and hepatic lipid features were assembled into an excel spreadsheet for each mouse in each of the 4 groups (RD, WDB, WDO, WDD). The data was analyzed using (<a href="http://www.metaboanalyst.ca/MetaboAnalyst/" target="_blank">http://www.metaboanalyst.ca/MetaboAnalyst/</a>) to create a heat map <b>[A]</b> and carry out a principal component analysis <b>[B]</b> and hierarchical clustering using Spearman’s ranked correlation <b>[C].</b> The heat map is a visualization of the changes in abundance/level of features for each animal. Animal identification numbers are listed on the right side of the heat map. The color ranges from deep orange (high abundance or level) to deep blue (low abundance or level); white represents no change.</p

Diet effects on hepatic morphology: Blockade arm.

<p>Livers sections from the 4 treatment groups (RD, WDB, WDO and WDD) in the Blockade Arm were stained with trichrome and photographed at 4x. Lipid droplets appear as white circles, while branching fibrosis appears as blue strands (yellow arrows) in the trichrome stained liver sections. The slides are representative of multiple sections of each liver and all livers in each group. PT, portal track; CV, central vein.</p

Analysis of hepatic fatty acids: Blockade arm.

<p>Hepatic fatty acids were quantified as described in Materials and Methods. <b>A:</b> The sum of fatty acids in the 4 lipid classes (saturated (SFA), monounsaturated (MUFA), ω3 and ω6 polyunsaturated fatty acids (PUFA) is presented in a stacked histogram. <b>B-D</b>: Specific fatty acids in each class are quantified and presented as μmol/mg protein. <b>E:</b> The mole ratio of the sum of ω3 and ω6 PUFA. Mean ± SD with 4–7. *, p<0.05 versus the RD group; #, p<0.05 versus the WDO group using one-way ANOVA.</p

Quantitation of plasma and hepatic osteopontin (Opn).

<p>Mouse plasma osteopontin levels were quantified by ELISA (R & D Systems). Results are expressed as Opn, ng/ml of plasma, N = 4–7; mean <u>+</u> SD; *, p<0.05 versus the RD group; #, p<0.05 versus the WDO group; one-way ANOVA. Hepatic Opn was quantified by immunoblot analysis of whole cell hepatic extracts as described previously. The mouse antibodies use in the analysis were anti-Opn (R & D Systems) and vinculin (Millipore); vinculin (Vin) was a loading control. Results were quantified by Licor Odyssey [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173376#pone.0173376.ref040" target="_blank">40</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173376#pone.0173376.ref060" target="_blank">60</a>] and expressed as abundance of Opn/Vin. Extracts from 3 separate livers in each group were examined by immunoblot analysis. Results are expressed as mean <u>+</u> SD; *, p<0.05 versus the RD group; #, p<0.05 versus the WDO group; one-way ANOVA.</p