Radioactive labeling experiments.

<p>[³⁵S]-L-methionine was used for metabolic labeling and subsequent immunoprecipitation of intracellular GDF-15 from total lysates (A) or secreted GDF-15 from cell culture medium (B). Cells were stimulated with TGF-ß1 for 4 h in (A) and 24 h in (B).</p

Reduced satiation to exogenous and endogenous CCK in DIO rats.

<p>A) CCK feeding study (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032967#s4" target="_blank">methods</a>) in Sprague Dawley rats after 8 weeks on respective diets. CCK8S (0.22 nmol/kg; i.p.) significantly inhibited food intake compared to vehicle in LF and DR, but not DIO rats. N = 6. B) CCK8S (2.19 nmol/kg; i.p.) significantly inhibited food intake in all rats. N = 6. C) Protocol for leptin feeding study in which endogenous CCK was upregulated D) Endogenous CCK reduced food intake in LF fed and DR rats at 4, 6 and 8 weeks. Endogenous CCK reduced food intake in DIO rats at 4 weeks but not at 6 and 8 weeks. N = 6; data expressed as mean ± SEM. Significant differences were represented as ** for p<0.01.</p

Expression analyses of GDF-15.

<p>(A) Upregulated transcript levels of GDF-15 in the presence of Dox using real-time qRT-PCR analysis. (B) Western blot analysis of GDF-15 protein expression in total protein lysates (30 μg) of TGF-ß1 stimulated cells (4 h). Expression of GDF-15 is increased when TGFBR2 is expressed (+Dox) in both cell clones but not in the parental Tet-On cell line. TGF-ß1 signaling is indicated by increased pSmad2 levels in the TGFBR2-expressing clones. Addition of cycloheximide (CHX) reduces the expression level of GDF-15. ß-Actin and Smad2 served as a loading control.</p

Effect of a high-fat (HF) diet on body weight and food intake <i>A</i>) Body weight of rats ingesting either a LF diet or HF diet (45% kcal) for 8 weeks.

<p>There was a significant increase in body weight in diet-induced obesity (DIO) rats compared with diet-induced obesity-resistant (DR) and LF animals. <i>B</i>) The increase in body weight of LF, DR, DIO rats expressed as a percent of weight gain from initial weight. DIO rats became significantly heavier than LF fed animals after 4 weeks of HF diet, and after 4 weeks of a HF diet compared to the DR rats. <i>C</i>) Average daily food intake (kcal) per week over the 8 weeks on the diets. In the first week, all rats on the HF diet had a significantly higher energy intake than LF fed rats but this initial hyperphagia only lasted for one week after which there was no significant difference in caloric intake between the groups. At <i>week 5</i>, the DIO group had a significantly higher energy intake than LF rats and DR rats. N = 12 per group; data expressed as mean ± SEM. Significant differences were represented as ^a,b,c between groups.</p

Supplemental Material - Characteristics of Patients With Psoriasis Treated With Various Biologics – A Danish Cohort Study

Supplemental Material for Characteristics of Patients With Psoriasis Treated With Various Biologics – A Danish Cohort Study by Christopher Willy Schwarz, Lone Skov, Alexander Egeberg, Alun Passey, Jennifer Lee, Patricia Gorecki, and Nikolai Loft in Journal of Psoriasis and Psoriatic Arthritis®</p

Transforming Growth Factor Beta Receptor 2 (TGFBR2) Changes Sialylation in the Microsatellite Unstable (MSI) Colorectal Cancer Cell Line HCT116

<div><p>Aberrant glycosylation is a common feature of many malignancies including colorectal cancers (CRCs). About 15% of CRC show the microsatellite instability (MSI) phenotype that is associated with a high frequency of biallelic frameshift mutations in the A10 coding mononucleotide microsatellite of the <i>transforming growth factor beta receptor 2</i> (<i>TGFBR2</i>) gene. If and how impaired TGFBR2 signaling in MSI CRC cells affects cell surface glycan pattern is largely unexplored. Here, we used the TGFBR2-deficient MSI colon carcinoma cell line HCT116 as a model system. Stable clones conferring doxycycline (dox)-inducible expression of a single copy wildtype <i>TGFBR2</i> transgene were generated by recombinase-mediated cassette exchange (RMCE). In two independent clones, dox-inducible expression of wildtype TGFBR2 protein and reconstitution of its signaling function was shown. Metabolic labeling experiments using the tritiated sialic acid precursor <i>N</i>-acetyl-D-mannosamine (ManNAc) revealed a significant decline (∼30%) of its incorporation into newly synthesized sialoglycoproteins in a TGFBR2-dependent manner. In particular, we detected a significant decrease of sialylated ß1-integrin upon reconstituted TGFBR2 signaling which did not influence ß1-integrin protein turnover. Notably, TGFBR2 reconstitution did not affect the transcript levels of any of the known human sialyltransferases when examined by real-time RT- PCR analysis. These results suggest that reconstituted TGFBR2 signaling in an isogenic MSI cell line model system can modulate sialylation of cell surface proteins like ß1-integrin. Moreover, our model system will be suitable to uncover the underlying molecular mechanisms of altered MSI tumor glycobiology.</p> </div

EGR-1 expression in VAN of DIO rats.

<p>A) Protein expression of EGR-1 in VANs of LF, DR and DIO rats treated with leptin. B) Densitometry analysis of <i>A</i> showing that EGR-1 levels in VAN are significantly reduced in DIO rats compared to LF and DR rats. N = 3. C) and D) Quantification of the number of EGR-1 immunopositive neurons in cultured VAN from DR rats (C) and DIO rats (D) fed a high fat diet for 8 weeks. C) Leptin reduced the concentration of CCK required to induce EGR-1 translocation in cultured VAN. D) Leptin had no effect on CCK induced EGR-1 translocation in cultured VAN. N = 6. Data expressed as mean ± SEM. Significant differences were represented as ^a,b,c between groups.</p

Altered neurochemical phenotype in DIO rats.

<p>Photomicrographs of sections of nodose ganglia to show immunoreactivity for Y2 (A), MCH1R (B), and CB1 (C) from fed and fasted Sprague Dawley rats after 8 weeks on respective diets. A) Y2 is barely detectable in nodose neurons of LF, DR, and DIO rats fasted 24 hours. Refeeding for 1 hour increased Y2 expression in LF and DR but not DIO rats. Quantification of positive Y2 cells as a percent of total cells. B) MCH1R and C) CB1 expression is elevated in fasted rats and is decreased by refeeding for 1 hour in LF and DR but not DIO rats. Quantification of positive MCH1R and CB1 cells as a percent of total cells. Representative images from experiments with four to six rats in each group are shown. Significant differences were represented as ^a,b,c between groups.</p

Altered CCK induced signaling in obese Zucker rats.

<p>Photomicrographs of sections of nodose ganglia to show immunoreactivity for Y2 (A), MCH1R (B), and CB1 (C) from lean and obese Zucker rats fasted 24 hr or refed for 1 hr. A) Y2 expression is elevated by feeding in lean Zucker rats, but not in obese Zucker rats. N = 4. B) MCH1R is decreased by feeding in lean Zucker rats but not in obese Zucker rats. N = 4. C) CB1 is decreased by feeding in lean and obese Zucker rats, but CB1 expression remains significantly higher in fed obese Zucker rats compared to lean Zucker rats. Significant differences were represented as ^a,b,c between groups.</p

Altered CCK induced satiety in obese Zucker rats.

<p>A) Obese Zucker rats weigh significantly more than lean Zucker rats (N = 12; t-test). B) Immunoblot of pSTAT3 in VAN of lean (top) and obese (bottom) Zucker rats treated with vehicle or leptin. C) Densitometry analysis of <i>B</i> showing that STAT3 is phosphorylated in response to leptin (i.p) in VAN of lean Zucker rats but not in obese Zucker rats. N = 4 D) Immunoblot of pSTAT3 in hypothalamus of lean and obese Zucker rats treated with leptin. E) Densitometry analysis of <i>D</i> showing that STAT3 is phosphorylated in response to leptin in the arcuate nucleus of the hypothalamus of lean Zucker rats, but not obese Zucker rats. (N = 4).F) CCK8S (i.p., 0.22 nmol/kg) significantly inhibited food intake in lean Zucker rats but not obese Zucker rats. N = 6. Data expressed as mean ± SEM. Significant differences were represented as ^a,b,c between groups in one-way ANOVA. Significant differences were represented as * for p<0.05; ** for p<0.01; and *** for p<0.001 in Student's t-test.</p