Frequencies of amino acid polymorphisms.

<p>Frequencies of amino acid polymorphisms.</p

blot analysis of PrP^C.

<p>Brain or spinal cord tissue was homogenized and analysed by Western blot as described in <i>Material & Methods</i> with various anti-PrP antibodies. Hooded Seal, one sample lanes 1–6; domestic dog, one sample lanes 7–8; domestic cat, two samples, 9–10 and 11–13; Brown bear, three samples 14–15, 16–17, 18–19. Untreated PrP^C: lanes 1,3,5,7,9,11,14,16,18. Deglycosylated PrP^C (+): lanes 2,4,6,8,10,12,13,15,17,19. Detection using antibodies with epitopes N-terminal to α-cleavage site: SAF32 (lanes 1–2) or F11 (lane 12), with epitope in α-cleavage site: 3F4 (lanes 3–4), with epitopes C-terminal to α-cleavage site: Bar224 (lanes 5–10, 14–19), 6H4 (lane 11) or BC6 (lane 13).</p

Particular sequence variations in PrP^C from species of the Order Carnivora.

<p>$ SEs: Sequence Elements, 9QG = nonapeptide PQGGGGWGQ, 9HA = nonapeptide PHAGGGWGQ, 8HG = octapeptide PHGGGWGQ, 9HG = nonapeptide PHGGGGWGQ; n, number of repeated SEs in brackets; r, number of total SEs (mode for all other species is five); Caniformia codon positions, equivalent codons for <i>Felidae</i> +3 when r = 5. (equivalent codons in <i>Primates</i> −4, in <i>Rodentia</i> −5). All other known <i>PRNP</i> sequences (>400): 98% N, 99% H, 99% K.</p

PrP protein sequence alignment.

<p>hyphen, identical amino acid to top sequence; dot, deletion; #, stop codon, end of coding region; L1–L8, cat lineages <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050623#pone.0050623-Hamir1" target="_blank">[34]</a>. White letters on black background, these positions are polymorphic (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050623#pone-0050623-t001" target="_blank">Table 1</a>). Sequences that are not shown as full length were generated with DNA primers on the end of the open reading frame. The correct species sequence can therefore not be inferred for these amino acids. Panthera (L1): Asiatic lion (Panthera leo persica), jaguar (Panthera onca), tiger (Panthera tigris & Panthera tigris sumatrae), Persian leopard (Panthera pardus saxicolor) Catopuma (L2): Asiatic golden cat (<i>Catopuma temminckii</i>) Leopardus (L4): margay (<i>Leopardus wiedii</i>) Puma (L6): cheetah (<i>Acinonyx jubatus</i>), mountain lion (<i>Puma concolor</i>) Felis (L8): cat (Felis catus), Bengal cat (Felis catus x Prionailurus bengalensis (L7)) Binturong: binturong (<i>Arctictis binturong</i>) Hyena: spotted hyena (<i>Crocuta crocuta</i>) Canis: coyote (Canis latrans), gray wolf (Canis lupus), dog (Canis familiaris) Vulpes: red fox (<i>Vulpes vulpes</i>) Speothos: bush dog (<i>Speothos venaticus</i>) Chrysocyon: maned wolf (<i>Chrysocyon brachyurus</i>) Ursus: brown bear (<i>Ursus arctos</i>), Black bear (<i>Ursus americanus</i>) Phoca: common seal (<i>Phoca vitulina</i>) Cystophora: hooded seal (<i>Cystophora cristata</i>) Pagophilus: harp seal (<i>Pagophilus groenlandica</i>) Otario: Patagonian sea lion (<i>Otaria flavescens</i>) Gulo: wolverine (<i>Gulo gulo</i>) Martes: European pine marten (<i>Martes martes</i>) Meles: badger (<i>Meles meles</i>) Mustela: European polecat (<i>Mustela putorius</i>) Lutra: European otter (<i>Lutra lutra</i>) Mephitis: striped skunk (<i>Mephitis mephitis</i>).</p

Knock down of NDRG1 expression by shRNAs.

<p>Western blots detect reduced NDRG1 expression in the breast cell lines ME16C2 and SUM102 expressing the shRNAs NDRG1si4 (si4) and NDRG1si7 (si7), transduced in parallel experiments (A and B) compared to control cells (C) transduced with the empty vector pSiRPG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087268#pone.0087268-Storvold1" target="_blank">[27]</a>. α-tubulin was used as loading control.</p

Increased NDRG1 expression due to hypoxia or ectopic expression.

<p>(<b>A</b>) Increased NDRG1 expression detected by Western blotting in the breast cell lines MCF-7 and ZR-75-1 grown at 1% O₂ for 24, 48 and 72 hours as indicated, compared to control cells grown at 20% O₂. (<b>B</b>) Western blots document increased NDRG1 expression in cells grown at 20% O₂. The ZR-75-1 cell populations transduced with the NDRG1-cDNA (six biological parallels indicated A–F), are compared to ZR-75-1 transduced with the vector pSiRPG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087268#pone.0087268-Storvold1" target="_blank">[27]</a> without insert (C2 and C3). α-tubulin was used as loading control (<b>A</b> and <b>B</b>). Note that a reduced level of total protein is loaded in the lane containing extract from MCF-7 grown at 1% O₂ for 24 hours. (<b>C</b>) Treeview presentation of the expression of 18 genes in a hypoxia signature gene list <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087268#pone.0087268-Chi1" target="_blank">[24]</a> in MCF-7 and ZR-75-1 cells grown at 1% O₂ for 24 or 48 hours as indicated. (<b>D</b>) Treeview presentation of the hypoxia signature genes in six ZR-75-1 cell populations ectopically over-expressing NDRG1 (indicated A–F). The red and green colours show increased and reduced expression levels, respectively (<b>C</b> and <b>D</b>). The colour intensity is from −3 to 3 indicating the magnitude of the fold change in gene expression between the cell population and its control.</p

Immunofluorescence staining of SUM102 cell cultures.

<p>Colors were applied artificially for graphical purposes. (<b>A</b>) NDRG1 (green) and calnexin (red) in untreated, hypoxia and doxorubicin treated cells. Bar: 5 µm. (<b>B</b>) NDRG1 (green) and EEA1 (red) in hypoxia treated cells. Bar: 10 µm.</p

PrP^C suppresses upregulation of <i>MX2</i> gene expression upon INF-α stimulation in SH-SY5Y cells.

<p>Untransfected human neuroblastoma SH-SY5Y cells and seven different clones transfected with a plasmid containing human <i>PRNP</i> to produce SH-SY5Y clones expressing human PrP^C, were stimulated for 3h with IFN-α (3 U/ml) (mean ± SEM, <i>n</i> = 4), and <i>MX2</i> gene expression was assessed. Six out of seven clones displayed a significantly lower response to IFN-α compared with the untransfected SH-SY5Y cells, using Dunnett’s post hoc test for multiple comparisons.</p

Expression of interferon-responsive genes in blood leukocytes after <i>in vivo</i> lipopolysaccharide (LPS) challenge in goats without PrP^C.

<p>RNA was extracted from circulating blood leukocytes from both genotypes, and gene expression was analyzed by FLUIDIGM qPCR. (A) Basal expression level (0 h) of selected interferon-responsive genes and <i>STAT1</i> in <i>PRNP</i>^+/+ (<i>n</i> = 12) and <i>PRNP</i>^Ter/Ter (<i>n</i> = 13) animals. (B) Gene expression of interferon-responsive genes and <i>STAT1</i> after <i>in vivo</i> LPS challenge (1 h) from <i>PRNP</i>^+/+ (<i>n</i> = 7) and <i>PRNP</i>^Ter/Ter (<i>n</i> = 8) animals. Values are mean ± SEM. Statistical significance is indicated by *, p-value < 0.05, as assessed by multiple t-tests.</p

Interferon-responsive genes dominate among the differentially expressed genes in goats lacking PrP^C.

<p>Graphical presentation of (A) the total number and percentage of differentially expressed genes (DEGs) between the two genotypes, compared to the average total number of genes expressed in peripheral blood mononuclear cells from both genotypes, and (B) the total number of upregulated and downregulated annotated DEGs. The fraction of upregulated (red) and downregulated (blue) interferon-responsive genes among the DEGs are also shown. (C) Hierarchical clustering of the interferon-responsive genes among the DEGs and expression data from all individual goats of both genotypes. Hierarchical clustering was performed using the ward algorithm on log2-normalized fold changes.</p