Cellular composition of tumors.

<p>Immunofluorescence images of OOC38-derived tumors grown in wild-type (n = 5) and <i>Fmod-/-</i> (n = 5) mice were used. Each of the markers was quantified as percentage of pixels per total number of pixels. Co-localization of NG2 and CD31 was quantified as percentage of co-localized pixels per total number of pixels. No differences between WT and <i>Fmod</i>-/- mice were observed (Mann-Whitney and Student’s <i>t</i>-test, p>0.05; error bars are standard deviations).</p

Properties of OOC38-derived carcinoma in wild-type and <i>Fmod-/-</i> mice.

<p>A) Masson Trichrome staining of OOC38 tumors grown in wild-type and <i>Fmod-/-</i> mice, depicting morphology and collagen fibers. B) Hydroxyproline content of OOC38 and KAT-4 tumors (WT n = 4, <i>Fmod</i>-/- n = 4), expressed as mg hydroxyproline/g tumor wet weight. C) Average number of days to first measurable tumor post cell inoculation (WT n = 18, <i>Fmod-/-</i> n = 32). D) Average number of days at which tumors reached an external measurement of 1 mm³ (WT n = 15, <i>Fmod</i>-/- n = 30). E) Ln values of externally measured tumor volumes (data points are means; error bars are standard deviations). Tumors were harvested within the indicated time frames (days post-injection: 24–30, 31–37, 38–44, 45–51). Number of tumors were, respectively: wild-type n = 8, 20, 33, 20; <i>Fmod -/-</i> n = 64, 51, 66, 47. No differences in tumor exponential growth were observed between WT and <i>Fmod</i>-/- (Student’s <i>t</i>-test, p>0.05).</p

Glycosaminoglycan (GAG) structure in OOC38 carcinomas grown in wild type and Fmod -/- mice (n = 4 + 4).

<p>Glycosaminoglycan (GAG) structure in OOC38 carcinomas grown in wild type and Fmod -/- mice (n = 4 + 4).</p

Gene expression (mRNA) data represented as the percent (%) of carcinoma grown in <i>Fmod-/-</i> compared to wild type mice.

<p>Gene expression (mRNA) data represented as the percent (%) of carcinoma grown in <i>Fmod-/-</i> compared to wild type mice.</p

Semi-quantitative assessment of decorin and biglycan.

<p>Dorsal skin proteins were extracted with guanidine- and dialyzed against urea-containing buffers. Total protein quantity was assessed by Bradford, and by gel imaging (A). Equal protein quantities were applied on anion-exchange beads and proteoglycans were eluted and imaged on gel (B); <i>Alb</i> is albumin control. Next, proteoglycan samples were digested with chondroitinase ABC and immunoblotted for decorin (C) and biglycan (D) to assess the quantities of the respective protein cores; <i>ND</i> is non-chondroitinase ABC-digested sample control (not visible in biglycan samples probably due to low quantity and smeary distribution).</p

Skin phenotype of <i>Aspn</i>^-/- mice.

<p>(A) Asporin immunoblotting of dorsal skin extracts from three wild-type and three <i>Aspn</i>^<i>-/-</i> mice. (B) 2 month-old female dorsal skins were analyzed histologically by staining with Massson Trichrome, or (C) by immunohistochemistry by staining for the presence of CD31-positive blood vessels and F4/80-positive macrophages; bars in A and <i>B</i> are 100 μm. Similar results were obtained from analyses of six mice from each genotype. (D) Transmission electron microscopy on cross-sectioned collagen fibrils in reticular dermis. (E) Transmission electron microscopy on longitudinally oriented collagen fibrils in reticular dermis. Bars in <i>C</i> and <i>D</i> are 200 nm. (F) Quantification of collagen fibril diameter in reticular dermis. 1,000 fibrils were measured from electron microscopy images collected from six wild-type and six <i>Aspn</i>^-/- mice.</p

<b>Human Genetic Variation at rs10071329 Correlates with Adiposity-related Traits, Modulates </b><b><i>PPARGC1B </i></b><b>Expression, and Alters Brown Adipocyte Function</b>

Human genetic variation in PPARGC1B has been associated with adiposity, but the genetic variants that affect PPARGC1B expression have not been experimentally determined. Here, guided by previous observational data, we used CRISPR/Cas9 to scarlessly edit the alleles of the candidate causal genetic variant rs10071329 in a human brown adipocyte cell line (hBAs). Switching the rs10071329 genotype from A/A to G/G enhanced PPARGC1B expression throughout the adipogenic differentiation, identifying rs10071329 as a cis-eQTL. The higher PPARGC1B expression in G/G cells coincided with greater accumulation of triglycerides, and higher expression of mitochondria-encoded genes, but without significant effects on adipogenic marker expression. Furthermore, G/G cells had improved basal- and norepinephrine-stimulated mitochondrial respiration, possibly relating to enhanced mitochondrial gene expression. The G/G cells also exhibited increased norepinephrine-stimulated glycerol release, indicating improved lipolysis. Altogether, our results showed that rs10071329 is a cis-eQTL, with the G/G genotype conferring enhanced PPARGC1B expression, with consequent improved mitochondrial function and response to norepinephrine in brown adipocytes. This genetic variant, and as yet undetermined eQTLs, at PPARGC1B could prove useful in genotype-based precision medicine for obesity treatment.</p

Quantification of gene transcripts using qPCR.

<p>Quantification of gene transcripts using qPCR.</p

Skin mechanical testing.

<p>Skin mechanical testing.</p

Skin glycosaminoglycan (GAG) analysis.

<p>GAGs were purified from two-month old dorsal skin (n = 5 per genotype) and digested with chondroitinase ABC or chondroitinase B or a mixture of heparinases. The resulting disaccharides were fluorescently labeled, separated by HPLC and quantified. (A) Total GAGs are the sum of chondroitin/dermatan sulfate (CS/DS), hyaluronic acid (HA), and heparan sulfate (HS). (B) Compositional analysis of CS/DS chains. UA = unsaturated hexuronic acid obtained after chondroitinases digestion, either unsulfated or 2-<i>O</i>-sulfated (UA-2S); GalNAc = <i>N</i>-acetyl-galactosamine either unsulfated or 4-<i>O</i>-sulfated (GalNAc-4S). (C) Compositional analysis of HS chains. The measured HS disaccharides were grouped according to the sulfation position: non-sulfated = UA-GlcNAc; <i>N</i>-sulfated = UA-GlcNS + UA-2S-GlcNS + UA-GlcNS-6S + UA-2S-GlcNS-6S; 2-<i>O</i>-sulfated = UA-2S-GlcNAc + UA-2S-GlcNAc-6S + UA-2S-GlcNS + UA-2S-GlcNS-6S; 6-<i>O</i>-sulfated = UA-GlcNAc-6S + UA-2S-GlcNAc-6S + UA-GlcNS-6S + UA-2S-GlcNS-6S. UA = unsaturated hexuronic acid obtained after heparinases digestion, either unsulfated or 2-<i>O</i>-sulfated (UA-2S); Glc = glucosamine either <i>N</i>-acetylated (GlcNAc) or <i>N</i>-sulfated (GlcNS) which can be both O-unsulfated or 6-<i>O</i>-sulfated (GlcNAc-6S and GlcNS-6S). Error bars show standard deviation.</p