Convergent Synthesis and Structural Confirmation of Phellodonin and Sarcodonin ε

The first synthesis of members of the sarcodonin family, phellodonin and sarcodonin ε, is reported herein. This verifies that the unprecedented and seemingly unstable <i>N</i>,<i>N</i>-dioxide-containing benzodioxazine framework can be constructed in the laboratory and lends further support to the proposed structures. The key step in the synthesis involves a biomimetic hetero-Diels–Alder reaction between a pyrazine <i>N</i>-oxide and an ortho-quinone

Convergent Synthesis and Structural Confirmation of Phellodonin and Sarcodonin ε

The first synthesis of members of the sarcodonin family, phellodonin and sarcodonin ε, is reported herein. This verifies that the unprecedented and seemingly unstable <i>N</i>,<i>N</i>-dioxide-containing benzodioxazine framework can be constructed in the laboratory and lends further support to the proposed structures. The key step in the synthesis involves a biomimetic hetero-Diels–Alder reaction between a pyrazine <i>N</i>-oxide and an ortho-quinone

Phosphoproteome Analysis of Formalin-Fixed and Paraffin-Embedded Tissue Sections Mounted on Microscope Slides

Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope slides are one of the largest available resources for retrospective research on various diseases, but quantitative phosphoproteome analysis of FFPE sections has never been achieved because of the extreme difficulty of procuring sufficient phosphopeptides from the limited amounts of proteins on the slides. Here, we present the first protocol for quantitative phosphoproteome analysis of FFPE sections by utilizing phase-transfer surfactant-aided extraction/tryptic digestion of FFPE proteins followed by high-recovery phosphopeptide enrichment via lactic acid-modified titania chromatography. We established that FFPE sections retain a similar phosphoproteome to fresh tissue specimens during storage for at least 9 months, confirming the utility of our method for evaluating phosphorylation profiles in various diseases. We also verified that chemical labeling based on reductive dimethylation of amino groups was feasible for quantitative phosphoproteome analysis of FFPE samples on slides. Furthermore, we improved the LC–MS sensitivity by miniaturizing nanoLC columns to 25 μm inner diameter. With this system, we could identify 1090 phosphopeptides from a single FFPE section obtained from a microscope slide, containing 25.2 ± 5.4 μg of proteins. This protocol should be useful for large-scale phosphoproteome analysis of archival FFPE slides, especially scarce samples from patients with rare diseases

Phosphoproteome Analysis of Formalin-Fixed and Paraffin-Embedded Tissue Sections Mounted on Microscope Slides

Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope slides are one of the largest available resources for retrospective research on various diseases, but quantitative phosphoproteome analysis of FFPE sections has never been achieved because of the extreme difficulty of procuring sufficient phosphopeptides from the limited amounts of proteins on the slides. Here, we present the first protocol for quantitative phosphoproteome analysis of FFPE sections by utilizing phase-transfer surfactant-aided extraction/tryptic digestion of FFPE proteins followed by high-recovery phosphopeptide enrichment via lactic acid-modified titania chromatography. We established that FFPE sections retain a similar phosphoproteome to fresh tissue specimens during storage for at least 9 months, confirming the utility of our method for evaluating phosphorylation profiles in various diseases. We also verified that chemical labeling based on reductive dimethylation of amino groups was feasible for quantitative phosphoproteome analysis of FFPE samples on slides. Furthermore, we improved the LC–MS sensitivity by miniaturizing nanoLC columns to 25 μm inner diameter. With this system, we could identify 1090 phosphopeptides from a single FFPE section obtained from a microscope slide, containing 25.2 ± 5.4 μg of proteins. This protocol should be useful for large-scale phosphoproteome analysis of archival FFPE slides, especially scarce samples from patients with rare diseases

In-depth Membrane Proteomic Study of Breast Cancer Tissues for the Generation of a Chromosome-based Protein List

The Chromosome-centric Human Proteome Project (C-HPP) aims to define all proteins encoded in each chromosome and especially to identify proteins that currently lack evidence by mass spectrometry. The C-HPP also prioritizes particular protein subsets such as membrane proteins, post-translational modifications, and low-abundance proteins. In this study, we aimed to generate deep profiling of the membrane proteins of human breast cancer tissues on a chromosome-by-chromosome basis using shotgun proteomics. We identified 7092 unique proteins using membrane fractions isolated from pooled breast cancer tissues with high confidence. A total of 3282 proteins were annotated as membrane proteins by Gene Ontology analysis, which covered 45% of the membrane proteins predicted in 20 859 protein-coding genes. Furthermore, we were able to identify 851 membrane proteins that currently lack evidence by mass spectrometry in neXtProt. Our results will contribute to the accomplishment of the primary goal of the C-HPP in identifying so-called “missing proteins” and generating a whole protein catalog for each chromosome

Effect of SK-216 on fibroblast to myofibroblast differentiation induced by TGF-β1 in human primary lung fibroblasts.

<p>After pre-incubation in serum free medium for 24 hours, ILD-derived and normal human primary lung fibroblasts were pretreated in the presence or absence of SK-216 (50, 150 μM) for 1 hour followed by stimulation with TGF-β1 (5 ng/ml). Real-time quantitative RT-PCR analysis and western blotting analysis were performed after incubation for 24 and 48 hours, respectively. (A) mRNA expression of α-SMA, fibronectin, and COL1A1 in ILD-derived human primary lung fibroblasts evaluated using real-time quantitative RT-PCR. Results are expressed as fold change from the control and reflect the mean ± SEM of 4 experiments. (B-C) Western blot analysis of α-SMA, fibronectin, and type I collagen in (B) ILD-derived and (C) normal human primary lung fibroblasts. Data normalized to β-actin levels are shown as fold change from the control and reflect the mean ± SEM of 3 independent experiments. *p <0.05, **p <0.01 vs untreated cells, ^#p <0.05, ^##p <0.01 vs cells treated with TGF-β1.</p

Effect of SK-216 on BLM-induced pulmonary fibrosis in mice.

<p>Mice were allocated into three groups: PBS+DW group, BLM+DW group, BLM+SK-216 group. Mice were intratracheally instilled with PBS or PBS containing BLM (1.5 mg/kg body weight) on day 0. PBS+DW group; PBS intratracheally instilled and orally administered with distilled water, BLM+DW group; BLM intratracheally instilled and orally administered with distilled water, BLM+SK-216 group; BLM intratracheally instilled and orally administered with distilled water on days from 0 to 8 and distilled water containing SK-216 (1000ppm) on days from 9 to 21. (A) mRNA expression levels of PAI-1, α-SMA, and COL1A1 in lung. Left lung harvested on day 11 was analyzed using real-time quantitative RT-PCR. Results are expressed as fold change from the control. (B) Levels of active PAI-1 and TGF-β1 in BALF. BALF was collected on day 11. (C) The degrees of pulmonary fibrosis were analyzed by measuring hydroxyproline contents in the whole lungs on day 21. (D) Histological and immunohistochemical analysis of bleomycin-injured lung. Right lungs were excised and sectioned on day 21. Scale bar = 200 μm. Results are presented as the mean ± SEM of 6 mice per group. *p <0.05, **p <0.01 vs PBS+DW group, ^#p <0.05, ^##p <0.01 vs BLM+DW group.</p

Additional file 6 of Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial–mesenchymal transition in lung endothelial cells

Additional file 6. Figure S5

Additional file 5 of Nestin and Notch3 collaboratively regulate angiogenesis, collagen production, and endothelial–mesenchymal transition in lung endothelial cells

Additional file 5. Figure S4

Effect of SK-216 on fibroblast to myofibroblast differentiation induced by TGF-β1 in MRC-5 cells.

<p>After pre-incubation in serum free medium for 24 hours, MRC-5 cells were pretreated in the presence or absence of SK-216 (50, 150 μM) for 1 hour followed by stimulation with TGF-β1 (5 ng/ml). Real-time quantitative RT-PCR analysis was performed after incubation for 24 hours. Proliferation assay, immunofluorescence staining, and western blotting analysis were performed after incubation for 48 hours. (A) Proliferation of MRC-5 cells after treatment with SK-216 and TGF-β1. (B) mRNA expression of α-SMA, fibronectin, and COL1A1 in MRC-5 cells evaluated using real-time quantitative RT-PCR. Results are expressed as fold change from the control and reflect the mean ± SEM of 4–5 experiments. (C) MRC-5 cells stained for α-SMA labeled with Zenon Alexa Fluor 488 (green) and nucleus labeled with DAPI (blue). Magnification: ×400. Scale bar = 50 μm. (D) Fluorescence intensities for α-SMA in MRC-5 cells. Results are expressed as Box plot of at least 6 fields. (E) Western blot analysis of α-SMA, fibronectin, and type I collagen. Data normalized to β-actin levels are shown as fold change from the control and reflect the mean ± SEM of 3 independent experiments. *p <0.05, **p <0.01 vs untreated cells, ^#p <0.05, ^##p <0.01 vs cells treated with TGF-β1.</p