Effect of purified bovine NPC2 on cholesterol accumulation in wild type and <i>NPC2^−/−</i> fibroblasts.

<p>(<b>A</b>) SDS-PAGE of 5 µg purified NPC2 resolved in a 10–20% gradient gel under nonreducing conditions and stained with Coomassie Brilliant Blue (<i>lane 1</i>). Molecular mass markers are shown on the left (<i>lane M</i>). (<b>B</b>) Upper row human- (Hu) and lower row murine (Mu) fibroblasts cultivated for 48 hours in complete medium (DMEM + 10% FBS). Wild type fibroblasts (<i>left panels</i>), <i>NPC2^-/-</i> fibroblasts (<i>middle panels</i>), <i>NPC2^-/-</i> fibroblasts supplemented with 600 nM NPC2 (<i>right panels</i>). Cells were fixed with 10% phosphate buffered formalin, pH 7.4 and stained with Filipin III and visualized using fluorescence microscope.</p

Immune response to NPC2 in 129P2 wild type- and <i>NPC2^−/−</i> mice.

<p>NPC2 coated microtiter wells were incubated with serial dilutions of immunized serum as indicated. Bound antibodies were detected by TRIFMA as described in Materials and Methods. An arbitrary concentration of anti-NPC2 antibodies was set to 1000 mU/ml in positive control serum prepared by subcutaneously injections of NPC2 with Freund's complete adjuvant as immune potentiator. Sera from saline treated healthy mice were similarly tested and served as negative controls. (<b>A</b>) Anti-NPC2 antibody concentration in intraperitoneal injected wild type mice, (<b>B</b>) Anti-NPC2 antibody concentration in intravenous injected wild type mice, and (<b>C</b>) Anti-NPC2 antibody concentration in intravenous NPC2 treated <i>NPC2^−/−</i> mice. Data represents mean values of triplicate wells.</p

Histochemical analysis of NPC2 replacement therapy in murine spleen sections.

<p>Hematoxylin<i>-</i>eosin (H&E) staining of spleen from saline treated wild type mice (<i>left panel</i>), saline treated <i>NPC2^−/−</i> mice (<i>middle panel</i>), and NPC2 treated <i>NPC2^−/−</i> mice (<i>right panel</i>). Massive accumulation of lipid droplets was most prominent observed in the spleens of saline treated NPC2<i>^−/−</i> mice. Data are representative of three separate experiments. <i>n</i> = 3 animals in each experimental group. Scale bares represent 100 µm.</p

Effect of NPC2 treatment on animal body weight.

<p>(<b>A</b>) Males (n = 4) and (<b>B</b>) females (n = 6), respectively, of saline-treated wild type mice (•), saline-treated <i>NPC2^−/−</i> mice (○), and NPC2 treated <i>NPC2^−/−</i> mice (▾). The mice were weighed weekly from P21 to P87. Each animal was injected twice weekly with saline or NPC2 (5 mg/kg). Values are means ± SEM.</p

Effect of NPC2 replacement therapy on murine brain cholesterol storage.

<p>Total cholesterol levels in cerebellum, cortex, and hippocampus was measured postmortem in saline treated wild type mice (<i>black bars</i>), saline treated <i>NPC2^−/−</i> mice (<i>light gray bars</i>), and NPC2 treated <i>NPC2^−/−</i> mice (<i>dark gray</i>). Each bar represents the mean ± SEM for 6 animals in each of the three groups. Bars not sharing a letter within a given panel are significantly different (<i>P</i><0.05).</p

Histochemical and immunohistochemical analysis of NPC2 replacement therapy in murine liver sections.

<p>Staining of representative tissue sections of 87 days old saline treated wild type mice (<i>left panels</i>), saline treated <i>NPC2^−/−</i> mice (<i>middle panels</i>), and NPC2 treated <i>NPC2^−/−</i> mice (<i>right panels</i>). Hematoxylin<i>-</i>eosin (H&E) staining (<i>first row</i>), immunohistochemical localisation of antigen F4/80 positive macrophages (brownish) (<i>second row</i>), and Masson's trichrome staining to detect collagen (blue) (<i>third row</i>). Lipid laden macrophages (Kupffer cells) are clearly visible and prominent in liver section from saline treated <i>NPC2^−/−</i> mice, whereas only a minority of the macrophages in liver sections from NPC2 treated <i>NPC2^−/−</i> mice are correspondingly loaded. Data are representative of three separate experiments. <i>n</i> = 3 animals in each experimental group. Scale bars represent 80 µm.</p

Generation and characterization of stable HCT116 cells with inducible miR-375 expression (HCT116-miR-375H).

<p>(A) Dox treatment induces increased expression of miR-375 in HCT116_miR-375H cells. The Relative expression of miR-375 was measured using RT-qPCR. The columns represent the mean of 3 replicates ± sd.*p-value<0.05 when compared to HCT116_ScrH cells. (B) xCELLigence real-time monitoring of cell proliferation. The cell index from time 12–96 hours is shown. Dox was added at time 0. (C) Dox treatment significantly reduces the proliferation of HCT116_miR-375H cells. Following the real-time monitoring in B, the slope (rate of changes in cell index) was calculated from time 60–80 hours (i.e. when changes in cell viability were apparent) and presented graphically. (D) Dox treatment specifically induces Caspase 3/7 dependent apoptosis in HCT116 miR-375H cells. The Caspase 3/7 activity was examined by fluorometric kinetic analysis and expressed relative to the Caspase 3/7 activity in untreated HCT116_ScrH cells. Z-DEVD-fmk (DEVD) was added to the cells six hours post-transfection. Data are presented as ±sd. of at least 2 independent experiments each with three biological replicates. *p-value<0.05. (E) Western blotting demonstrating down-regulation of YAP1 in dox treated HCT116_miR-375H cells compared to non-treated and HCT116_ScrH cells. Loading control: β-actin. (F) miR-375 expression reduces tumor growth <i>in vivo</i>. Growth curves of tumors generated in nude mice injected with HCT116_miR-375H cells treated with (n = 4) or without (n = 4) dox in the drinking water. Dox was added to the drinking water when the tumor size was >50 mm³. Data marks and bars represent the mean ±sd. *p-value<0.05.</p

Model of the role of miR-375 in the regulation of apoptotic death.

<p>Model of the role of miR-375 in the regulation of apoptotic death.</p

The most differentially expressed miRNAs in stage II colorectal adenocarcinomas and their ability to induce phenotypic changes in the high-throughput analysis.

1<p>A FC _(log2)≤−1.50 and ≥1.50 and a p-value≤0.01 was considered significant (Mann-Whitney U test).</p><p>NA: miRNAs not included in the pre-miRNA library from Ambion.</p><p>+: miRNAs that induced phenotypic changes (Top-40 ranked).</p><p>(+): miRNAs that induced phenotypic changes in at least one cell line (not Top-40 ranked).</p><p>A: Induction of apoptosis.</p><p>P: Inhibition of proliferation.</p

Expression of known direct miR-375 targets.

Δ<p>Analyzed in 24 normal mucosa and 30 MSS adenocarcinomas that has previously been profiled using Human Exon 1.0 ST arrays (Thorsen K et al. Alternative Splicing of SLC39A14 in Colorectal Cancer is Regulated by the Wnt Pathway,</p><p>Molecular and Cellular Proteomics, 2011).</p><p>ND: not detected (median log intensity <7).</p><p>NS: not significant (a p-value≤0.05 was considered significant).</p>‡<p>: not present on the Human Gene 1.0ST.</p><p>*Hs: Homo sapiens.</p>¤<p>Mm: Mus musculus.</p>∧<p>The probes on the Human Gene 1.0ST arrays recognize more than one transcript.</p