Additional file 1: of Acid ceramidase deficiency: Farber disease and SMA-PME

A description of the literature search method used to identify FD and SMA-PME patients from research articles and case reports. (DOCX 20 kb

Neutral sphingolipid percent distribution in NPD Tre cells pre-incubated with the indicated concentrations of NDJ or PDMP.

<p>Lipids were separated by HPTLC and their concentrations were determined after acid hydrolysis using fluorescamine as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019974#pone.0019974-Kisic1" target="_blank">[37]</a>.</p

Effects of IL-6 deficiency on liver lipid content.

<p>Lipids were extracted from the livers of mice fed a normal or the MCD diet for 5 weeks. Phosphatidylcholine (PC, panel A) and phosphatidylethanolamine (PE, panel B) levels were measured by HPLC, and the PC/PE ratio (C) was calculated. The levels of sphingomyelin (D) were determined by inorganic phosphorus analysis preceded of alkaline methanolysis, and those of ceramide (E) were measured by the diacylglycerol kinase assay. In panel F, ceramide is expressed as the ratio to total phospholipids. Data are expressed as in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007929#pone-0007929-g001" target="_blank">Fig. 1</a>. Further symbols ($p<0.05,$$p<0.01,$$$ p<0.001) indicate differences between IL-6^−/− and WT mice on a normal diet.</p

Thin-layer chromatography immunostaining with a mixture of anti-GM3, anti-GM2 and anti-GM1 Mabs of gangliosides of Tre and MS1418+ cell lines after 10 min treatment with anti-Fas.

<p>Std represents standard gangliosides from bovine brain. The amounts of gangliosides applied on the HPTLC plate were one tenth of those visualized by resorcinol-HCl in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019974#pone-0019974-g001" target="_blank">Fig. 1<i>a</i></a>.</p

MCD diet results in loss of body and liver weights, hypoglycemia, hypotriglyceridemia, hypocholesterolemia, and increased serum alanine aminotransferase (ALT) activity.

<p>After 5 weeks of a MCD diet, body (A) and liver (B) weights, serum levels of glucose (C), triacylglycerols (D), cholesterol (E), and ALT (F) of WT and IL-6^−/− mice were determined. Data are means±SEM; the number of animals is indicated into each column. Asterisks denote significant differences (* p<0.05, ** p<0.01, *** p<0.001) between mice of different genotypes on the MCD diet (when indicated above the line) or between different diets for the same genotype (when indicated in A and above the columns). $$$ indicates a difference (p<0.001) between IL-6^−/− and WT mice on a normal diet.</p

Sequences of primers used for quantitative RT-PCR.

<p>All sequences are oriented, from left to right, 5′ to 3′.</p

Effect of IL-6 deficiency on MCD diet-induced steatohepatitis.

<p>After 5 weeks of a MCD diet, mice were sacrificed and the liver was examined for scoring steatosis (A), ballooning (not shown) and lobular inflammation (B). The NAFL (non-alcoholic fatty liver) activity score was calculated (C). Representative sections of hematoxylin/eosin-stained liver of WT mice on normal (D) or MCD diet (E), or of IL-6^−/− mice on MCD diet (F), are shown. Liver sections of WT mice (E) revealed large fat droplets and the presence of inflammatory cells. In IL-6^−/− mice, steatosis was similarly present, but the number of inflammatory cells was decreased. The number of animals per group was 12 and 11 for WT mice, and 10 and 10 for IL-6^−/−mice fed a normal or MCD diet, respectively. * p<0.05, *** p<0.001.</p

Pharmacological inhibition of ganglioside production does not prevent from anti-Fas-induced apoptosis.

<p>(<i>a</i>) Production of gangliosides in control lymphoblasts after treatment with 200 ng/ml anti-Fas antibody for 10 min with or without pre-treatment with 10 µM PDMP. Melanoma (Mel. Std) and bovine brain (Brain Std) ganglioside mixtures were used as standards. (<i>b</i>) Effect of PDMP on the production of gangliosides by NPD (MS1418) cells that were incubated as described in (<i>a</i>). (<i>c</i>) Effect of PDMP on the production of neutral glycosphingolipids by NPD (MS1418) cells that were incubated as described in (<i>a</i>). GlcCer, glucosylceramide; LacCer, lactosylceramide. (<i>d</i>) Control (Con) and NPD lymphoblasts were pre-incubated for 24 h with or without 10 µM PDMP or 400 µM DNJ, and treated without or with 100 ng/ml anti-Fas antibody for 5 h under serum-free conditions. Cell surface exposure of phosphatidylserine was determined by flow cytometry using annexin V-FITC/PI labelling (ns, not significant). (<i>e</i>) Control (Con), NPD (MS1418) and aSMase-transduced NPD (MS1418+) cells were incubated as indicated in (<i>d</i>) and cell extracts were subjected to 15% SDS-PAGE and immunoblotted with anti-caspase-3 antibody. Migrations indicated are: pro-caspase-3, full-length inactive caspase-3; p20 and p17, active subunits.</p

Schematic view of ganglioside biosynthesis in mammalian cells.

<p>Sequential addition of sialic acid residues to lactosylceramide (LacCer) by the specific sialyl-transferases I, II and III produces the monosialo, disialo and trisialo-gangliosides GM3, GD3 and GT3, respectively. These glycolipids serve as precursors for the a, b and c series of gangliosides, which are formed by stepwise glycosylation, i.e., by addition of N-acetyl-beta-galactosamine (GalNAc), beta-galactose (Gal) and sialic acid by a GalNAc-transferase, a galactosyl-transferase and a sialyl-transferase, respectively.</p

Effect of IL-6 deficiency on Kupffer cell activation during MCD diet-induced steatohepatitis.

<p>Representative pictures of immunohistochemical detection of F4/80-positive cells in the liver of WT and IL-6^−/− mice on normal (A and C) or MCD diet (B and D), respectively, are shown. Arrowheads indicate isolated Kupffer cells whereas arrows show macrophage clusters. The abundance of isolated cells and clusters was estimated in 12 and 11 livers for WT mice, and 10 and 10 for IL-6^−/− mice fed a normal or MCD diet, respectively (** p<0.01, *** p<0.001 when comparing different diets for the same genotype).</p