Effects of adiponectin on lipid production in human sebocytes.

<p>(A) Detection of intracellular lipids in sebocytes treated with adiponectin using microscopy after Oil Red O and Nile red staining. Scale bars = 20 μm. (B) Lipid levels in sebocytes treated with various doses of adiponectin, calculated as percentages of the value of untreated cells. (C) Relative abundance of major lipid classes determined by thin-layer chromatography. Human sebocytes grown in the presence of [¹⁴C]-acetate after treatment with adiponectin, and changes in specific lipid components such as cholesterol, triglyceride, wax ester, and squalene, were analyzed. (D) The effect of adiponectin in three-dimensional (3D) culture of human sebocytes. Sebocytes in 400 μL of medium were added to the matrigel layer (50–100 μL) adding to glass-bottom dishes. Sebocytes were treated with 200 ng/mL adiponectin and the overlay medium was replaced every 2 days during culture of the cells for 7 days. Organoid structures in 3D culture of sebocytes were stained with hematoxylin and eosin (H&E), Nile red, SREBP and epithelial membrane antigen (EMA). Scale bars = 20 μm. Data represent means ± SEM (<i>n</i> = 8). Data were analyzed using Student’s <i>t</i> test (*<i>P</i> < 0.05, **<i>P</i> < 0.01).</p

Expression of adiponectin receptors in human sebaceous glands and sebocytes.

<p>(A) Human sebaceous glands were assayed for adiponectin receptor (AdipoR)1 and AdipoR2 by immunohistochemistry. Inset, isotype control. (B) Immunofluorescence labeling of AdipoR1 and AdipoR2 (green) in human sebocytes. Nuclei were counterstained with DAPI (blue). Inset, isotype control. (C, D) Western blotting and RT-PCR of sebocyte lysates. Human keratinocytes and fibroblasts expressing AdipoR1 and AdipoR2 were used as positive controls. Scale bars = 20 μm. Sebo, sebocytes; KC, keratinocytes; FB, fibroblasts.</p

Revising a Personal Genome by Comparing and Combining Data from Two Different Sequencing Platforms

<div><p>For the robust practice of genomic medicine, sequencing results must be compatible, regardless of the sequencing technologies and algorithms used. Presently, genome sequencing is still an imprecise science and is complicated by differences in the chemistry, coverage, alignment, and variant-calling algorithms. We identified ∼3.33 million single nucleotide variants (SNVs) and ∼3.62 million SNVs in the SJK genome using SOLiD and Illumina data, respectively. Approximately 3 million SNVs were concordant between the two platforms while 68,532 SNVs were discordant; 219,616 SNVs were SOLiD-specific and 516,080 SNVs were Illumina-specific (<i>i</i>.<i>e</i>., platform-specific). Concordant, discordant, and platform-specific SNVs were further analyzed and characterized. Overall, a large portion of heterozygous SNVs that were discordant with genotyping calls of single nucleotide polymorphism chips were highly confident. Approximately 70% of the platform-specific SNVs were located in regions containing repetitive sequences. Such platform-specificity may arise from differences between platforms, with regard to read length (36 bp and 72 bp vs. 50 bp), insert size (∼100–300 bp vs. ∼1–2 kb), sequencing chemistry (sequencing-by-synthesis using single nucleotides vs. ligation-based sequencing using oligomers), and sequencing quality. When data from the two platforms were merged for variant calling, the proportion of callable regions of the reference genome increased to 99.66%, which was 1.43% higher than the average callability of the two platforms, representing ∼40 million bases. In this study, we compared the differences in sequencing results between two sequencing platforms. Approximately 90% of the SNVs were concordant between the two platforms, yet ∼10% of the SNVs were either discordant or platform-specific, indicating that each platform had its own strengths and weaknesses. When data from the two platforms were merged, both the overall callability of the reference genome and the overall accuracy of the SNVs improved, demonstrating the likelihood that a re-sequenced genome can be revised using complementary data.</p> </div

Chip-concordance of callable and non-callable regions.

<p>Chip-concordance of callable and non-callable regions.</p

Cumulative frequency plot of sequencing depths in heterozygous calls. The sequencing depths of heterozygous calls in the SOLiD data are plotted.

<p>The patterns of concordant SNVs that are either chip-concordant or chip-discordant are almost compatible, which explains why the majority of heterozygous concordant SNVs that are chip-concordant are highly confident calls. In contrast, the median of discordant SNVs that are chip-discordant is substantially lower than those of concordant SNVs, which explains why only 25% of them are highly confident calls.</p

Concordance of SNVs identified by the two different sequencing platforms.

<p>Concordance of SNVs identified by the two different sequencing platforms.</p

Patterns of chip-discordance in concordant SNVs between platforms.

<p>Patterns of chip-discordance in concordant SNVs between platforms.</p

<i>Traf6</i>-deficient primary cells show a higher sensitivity to TGF-β1.

<p><i>Traf6</i>-deficient primary cells show a higher sensitivity to TGF-β1.</p

TRAF6 forms a complex with TβRIII in response to TGF-β1.

<p>(<b>A</b>) CAGA12-Luciferase assays were performed in HepG2 cells. The plasmids encoding HA-TβRIII, TRAF6, CAGA12-Luc, and Renilla-luc reporter gene were transfected as indicated and, on the next day, TGF-β1 (0.4 ng/ml) and/or IL-1β (20 ng/ml) was added for 16 hours. The obtained relative luciferase units(RLU) were normalized by renilla luciferase activities. (<b>B</b>) Using the control and TβRIII knock-down HaCaT cells, TGF-β1 (0.4 ng/ml) and/or IL-1β (20 ng/ml) were treated as shown for up to 3 hours. The level of total Smad3 and phospho-Smad3 protein was detected by immunoblotting. For the control of equal loading, β-actin was used. (<b>C</b>) HEK293 cells stably expressing HA-TβRIII were transfected with Myc-Traf6 plasmids and then treated with TGF-β1. Cells were harvested at various times and were subjected to immunoprecipitation with anti-HA antibody. Co-immunoprecipitated TRAF6 was detected with anti Myc antibody. (<b>D</b>) Complex formation ability between TβRIII and TRAF6 wild-type or the TRAF6 (C85A/H87A) E3 ligase mutant was compared after TGF-β stimulation for an hour. (<b>E</b>) According to the manufacturer's protocol, interaction was visualized by <i>in situ</i> proximity ligation assay (O-link) with proximity probes directed against TRAF6 and TβRIII using Alexa 555 labeling (red). Endogenous TRAF6 was co-localized with HA-TβRIII along the plasma membrane in the presence of TGF-β (red blobs). Bar = 2.5 µm. (<b>F</b>) Quantification of blobs per cell was carried out by semi-automated image analysis using the freeware software BlobFinder V 3.0. (<b>G</b>) HA-TβRIII-stably expressing 4T07 mouse mammary cancer cells were treated with TGF-β and LPS for one hour. Co-immunoprecipitation was carried out using anti-HA antibody to query interaction with endogenous TRAF6. The results are representative of three independent experiments.</p

TRAF6 mediates IL-1β or LPS-induced suppression of TGF-β1/Smad pathway.

<p>(<b>A</b>) HEK293 cells were treated with TGF-β1 (0.4 ng/ml) and/or IL-1β (2 ng/ml) as indicated. TGF-β-mediated Smad3 phosphorylation was demonstrated by anti-pSmad3 and total Smad3 antibodies. As a loading control, α-tubulin was used. (<b>B</b>) SBE-Luc assay was performed in HepG2 cells. These luciferase assays were normalized by the activities of co-transfected β-galactosidase. (<b>C</b>) TRAF6 or GFP was over-expressed in HEK293 cells by use of a lentiviral system. Cells were harvested after TGF-β1 addition for up to 6 hours followed by westernblotting to compare phospho-Smad2/3 levels. (<b>D</b>) TGF-β1 target genes, <i>CDKN2B</i>, <i>CDKN1A</i>, and <i>SMAD6</i>, were detected by quantitative RT-PCR using total RNA from vector-(GFP) or TRAF6-expressing HaCaT cells treated as indicated. Human <i>GAPDH</i> was used as a loading control. (<b>E</b>) FaO cells were infected with either control vector or Myc-TRAF6 on previous day and then treated with TGF-β alone or together with LPS up to 8 hours. Both floating and adherent cells were harvested to compare the induction of cleaved caspase-3. TGF-β-induced signal transduction was displayed by showing pSmad2 level. The results are representative of three independent experiments.</p