Figure 1
Brief summary of GeLC-MS/MS and bioinformatics methodology. GeLC-MS/MS, gel liquid chromatography-tandem mass spectrometry
Figure 2
Sirius Red staining of (A) control, (B) TAA, (C) TAA + AM groups and their respective threshold image for the quantification of percent area stained red. AM, α-mangostin; TAA, thioacetamide. Scale bars in blue and red represent 100 μm
Figure 3
Automated quantification of Sirius Red staining using ImageJ across the 3 experimental groups plotted as relative area (%) stained red. Bars indicate the means and error bars represent standard deviation (SD). ***P < 0.001 when the TAA group (mean ± SD; 4.86 ± 0.56%) was compared with the control (1.37 ± 0.55%) and TAA + AM group (2.50 ± 0.81%). **P < 0.01 (= 0.002) when the TAA + AM group (2.50 ± 0.81%) was compared with the control group AM, α-mangostin; TAA, thioacetamide
Table 1
Proteins correlated with quantified Sirius Red collagen staining identified by using the self-organizing tree algorithm of Mev software (version 4.6.1). We identified 68 proteins. Accession numbers searchable on NCBI and their identity details are tabulated. P-values from t tests are also tabulated to indicate the confidence of identification using Mascot software. The numbers in the column for each group represent the log2 ion peak intensity for each species.
| NCBI accession | Identity details | †P | Control | TAA | TAA+AM |
|---|---|---|---|---|---|
| NP_036894.2 | Caspase-1 | 7.17E–03 | 1.59E+01 | 1.62E+01 | 1.55E+01 |
| NP_001100608.1 | Protein strawberry notch homolog 1 | 4.09E–04 | 0.00E+00 | 1.74E+01 | 0.00E+00 |
| NP_001005905.1 | T-complex protein 1 subunit beta | 5.40E–03 | 0.00E+00 | 1.75E+01 | 0.00E+00 |
| NP_001020151.1 | Ankyrin repeat domain-containing protein 34A | 3.69E–03 | 1.67E+01 | 1.73E+01 | 1.65E+01 |
| AAB82191.1 | Anti-fluorescein monoclonal IgM heavy chain | 5.22E–07 | 1.59E+01 | 1.67E+01 | 1.49E+01 |
| NP_037081.2 | Beta-enolase | 6.56E–03 | 2.05E+01 | 2.12E+01 | 1.95E+01 |
| NP_113948.1 | Bile salt export pump | 5.84E–03 | 1.91E+01 | 1.96E+01 | 1.76E+01 |
| AAI66492.1 | Bms1 protein | 6.05E–03 | 1.59E+01 | 1.62E+01 | 1.55E+01 |
| NP_058801.1 | Bone morphogenetic protein 3 precursor | 7.67E–03 | 1.95E+01 | 2.16E+01 | 1.86E+01 |
| AAH98803.1 | C1r protein | 8.13E–04 | 1.82E+01 | 1.95E+01 | 1.85E+01 |
| NP_001029036.1 | Cflar isoform 1‡ | 1.38E–06 | 2.06E+01 | 2.06E+01 | 2.05E+01 |
| NP_001099374.1 | CD209a molecule | 6.01E–03 | 1.34E+01 | 1.61E+01 | 1.43E+01 |
| NP_001102339.1 | Cell death activator CIDE-B | 8.09E–03 | 0.00E+00 | 1.92E+01 | 0.00E+00 |
| NP_001094143.1 | Mapk8ip3‡ | 1.61E–04 | 1.64E+01 | 1.67E+01 | 1.54E+01 |
| AAL86014.1 | Co-activator/regulatory zinc-finger protein NIF | 2.32E–03 | 1.85E+01 | 1.92E+01 | 1.80E+01 |
| NP_690059.1 | Coagulation factor VII precursor | 3.99E–03 | 1.88E+01 | 1.97E+01 | 1.85E+01 |
| NP_001137368.1 | Coiled-coil domain-containing protein 137 | 6.79E–04 | 1.89E+01 | 1.95E+01 | 1.88E+01 |
| NP_001004262.1 | Conserved oligomeric Golgi complex subunit 6 | 7.10E–03 | 1.85E+01 | 1.96E+01 | 1.76E+01 |
| NP_598218.1 | CX3C chemokine receptor 1 | 8.30E–03 | 1.71E+01 | 1.83E+01 | 1.73E+01 |
| NP_001013155.1 | C-X-C motif chemokine 14 precursor | 3.64E–03 | 1.66E+01 | 1.81E+01 | 1.69E+01 |
| NP_001013201.1 | Cytosolic Fe-S cluster assembly factor NARFL | 2.90E–03 | 1.44E+01 | 1.68E+01 | 1.45E+01 |
| NP_001129274.1 | Disks large-associated protein 5 | 8.75E–03 | 1.52E+01 | 1.63E+01 | 1.51E+01 |
| NP_001102999.1 | DNA/RNA-binding protein KIN17 | 7.11E–03 | 1.98E+01 | 2.08E+01 | 1.85E+01 |
| NP_064311.2 | E3 ubiquitin-protein ligase ARIH1 | 5.07E–03 | 1.59E+01 | 1.63E+01 | 1.49E+01 |
| NP_620252.1 | Gamma-aminobutyric acid receptor subunit rho-3 precursor | 2.44E–03 | 1.40E+01 | 1.58E+01 | 1.40E+01 |
| NP_036702.1 | Glutamate dehydrogenase 1, mitochondrial precursor | 4.81E–03 | 1.64E+01 | 1.85E+01 | 1.71E+01 |
| NP_899653.2 | Glutathione peroxidase 2 | 7.58E–03 | 0.00E+00 | 1.64E+01 | 0.00E+00 |
| NP_001007636.2 | GTPase activating protein testicular GAP1 | 3.01E–03 | 1.19E+01 | 1.28E+01 | 1.04E+01 |
| NP_001101888.1 | HEAT repeat-containing protein 1 | 5.38E–03 | 1.82E+01 | 2.04E+01 | 1.84E+01 |
| NP_001099862.1 | Heparan sulfate glucosamine 3-O-sulfotransferase 5 | 3.94E–03 | 1.65E+01 | 1.85E+01 | 1.66E+01 |
| NP_001099302.1 | Homeobox protein DLX-3a | 7.95E–04 | 1.67E+01 | 1.79E+01 | 1.67E+01 |
| NP_001102748.1 | Iron-sulfur cluster assembly 2 homolog, mitochondrial precursor | 1.56E–04 | 1.57E+01 | 1.72E+01 | 1.47E+01 |
| ACX55121.1 | Jarid1d protein | 9.36E–03 | 1.43E+01 | 1.67E+01 | 1.41E+01 |
| NP_001102270.1 | Krueppel-like factor 7 | 6.68E–04 | 1.62E+01 | 1.76E+01 | 1.66E+01 |
| NP_001101180.1 | Leucine-rich repeats and immunoglobulin-like domains protein 2 precursor | 2.94E–04 | 1.87E+01 | 1.93E+01 | 1.81E+01 |
| CAA62024.1 | Mature alpha chain of major histocompatibility complex class I antigen | 2.91E–03 | 1.67E+01 | 1.76E+01 | 1.58E+01 |
| AFD32168.1 | Mitogen-activated protein kinase kinase kinase 1 | 5.26E–03 | 1.51E+01 | 1.66E+01 | 1.50E+01 |
| NP_001101771.1 | Map3k14‡ | 9.93E–04 | 0.00E+00 | 2.12E+01 | 0.00E+00 |
| AAN86532.1 | Multidrug resistance-associated protein 1 | 2.53E–06 | 1.53E+01 | 1.60E+01 | 1.44E+01 |
| NP_446266.2 | Myosin phosphatase Rho-interacting protein isoform 1 | 1.34E–04 | 1.36E+01 | 1.62E+01 | 1.45E+01 |
| NP_663707.1 | N-acetyllactosaminide alpha-1,3-galactosyltransferase | 5.28E–03 | 1.84E+01 | 1.93E+01 | 1.87E+01 |
| NP_001000033.1 | Olfactory receptor Olr1507 | 4.95E–03 | 1.65E+01 | 1.66E+01 | 1.61E+01 |
| NP_001000682.1 | Olfactory receptor Olr486 | 6.49E–04 | 1.64E+01 | 1.66E+01 | 1.59E+01 |
| NP_001005384.1 | Oncostatin-M-specific receptor subunit beta precursor | 2.38E–04 | 1.76E+01 | 1.92E+01 | 1.61E+01 |
| AAI69002.1 | Phosphatase, orphan 1 | 7.90E–03 | 1.92E+01 | 2.08E+01 | 1.94E+01 |
| NP_114007.1 | Protocadherin Fat 1 precursor | 4.82E–04 | 0.00E+00 | 1.71E+01 | 0.00E+00 |
| NP_536320.1 | Puromycin-sensitive aminopeptidase precursor | 7.38E–05 | 1.56E+01 | 1.71E+01 | 1.60E+01 |
| NP_001008376.1 | Lamtor3‡ | 3.60E–04 | 1.58E+01 | 1.64E+01 | 1.50E+01 |
| NP_001103357.1 | Receptor-type tyrosine-protein phosphatase C isoform 3 precursor | 1.62E–03 | 1.72E+01 | 1.94E+01 | 1.65E+01 |
| P38650.1 | Cytoplasmic dynein 1 heavy chain 1 | 6.45E–03 | 1.76E+01 | 1.92E+01 | 1.77E+01 |
| AGG39759.1 | Rh-associated glycoprotein, partial | 1.93E–03 | 1.63E+01 | 1.67E+01 | 1.63E+01 |
| NP_001161999.1 | Selenocysteine insertion sequence-binding protein 2-like | 7.31E–03 | 1.52E+01 | 1.63E+01 | 1.55E+01 |
| NP_579826.1 | Seminal vesicle antigen-like 1 precursor | 1.50E–04 | 1.96E+01 | 1.99E+01 | 1.87E+01 |
| NP_954526.1 | Seminal vesicle secretory protein 1 precursor | 3.13E–04 | 1.57E+01 | 1.60E+01 | 1.55E+01 |
| AAL77056.1 | SH2 phosphatase 1 | 4.31E–03 | 1.88E+01 | 2.01E+01 | 1.93E+01 |
| NP_001041645.1 | SH2B adapter protein 1 isoform 2 | 5.69E–04 | 1.79E+01 | 1.85E+01 | 1.81E+01 |
| EDM04919.1 | Similar to G protein pathway suppressor 2, isoform CRA_d | 2.32E–03 | 1.60E+01 | 1.81E+01 | 1.61E+01 |
| EDM18475.1 | Similar to RIKEN cDNA 1810020D17, isoform CRA_c | 6.74E–03 | 0.00E+00 | 1.74E+01 | 0.00E+00 |
| NP_113871.1 | Structural maintenance of chromosomes protein 1A | 5.12E–03 | 1.76E+01 | 1.84E+01 | 1.73E+01 |
| CAA33552.1 | Sucrase isomaltase, partial | 6.23E–03 | 1.61E+01 | 1.66E+01 | 1.62E+01 |
| CAC37104.1 | TA1 KET beta protein | 7.79E–03 | 1.68E+01 | 1.76E+01 | 1.63E+01 |
| BAH79733.1 | Tensin3 | 1.82E–03 | 1.63E+01 | 1.68E+01 | 1.65E+01 |
| AAI60846.1 | Terf2 protein | 4.22E–05 | 0.00E+00 | 1.78E+01 | 0.00E+00 |
| NP_001258196.1 | Transcription factor SOX-5 | 2.84E–04 | 1.59E+01 | 1.61E+01 | 1.55E+01 |
| NP_620221.2 | Translation initiation factor eIF-2B subunit epsilon | 4.87E–05 | 1.42E+01 | 1.45E+01 | 1.36E+01 |
| NP_001102110.2 | Tubulin monoglycylase TTLL3 | 2.82E–07 | 0.00E+00 | 1.68E+01 | 0.00E+00 |
| NP_001100418.1 | Ubiquitin carboxyl-terminal hydrolase 26 | 8.37E–03 | 1.78E+01 | 1.86E+01 | 1.80E+01 |
| NP_598280.1 | UDP-glucose:glycoprotein glucosyltransferase 1 precursor | 8.43E–03 | 1.95E+01 | 2.00E+01 | 1.84E+01 |
[i] †t test.
‡Four input proteins were found to be extensively linked to 4 main pathways for fibrosis drawn from the KEGG pathway database upon analysis by STITCH 5.0. Cflar, CASP8 and FADD-like apoptosis regulator; Lamtor3, Ragulator complex protein LAMTOR3; KEGG, Kyoto Encyclopedia of Genes and Genomes; Mapk8ip3, C-Jun-amino-terminal kinase-interacting protein 3; AM, a-mangostin; Map3k14, Mitogen-activated protein kinase kinase kinase 14; NCBI, National Center for Biotechnology Information, U.S. National Library of Medicine; TAA, thioacetamide
Figure 4
Cflar protein (red node) and its functional partners upon analysis by STITCH 5.0. Analysis of the KEGG identified that Cflar is directly involved pathways corresponding to apoptosis, TNF signaling, NK-κB signaling, and Chagas disease (American trypanosomiasis). Uniprot accession: C0H5Y5, Mass: 55,059 Da, Cellular location: Cytosol. Cflar, CASP8 and FADD-like apoptosis regulator; KEGG, Kyoto Encyclopedia of Genes and Genomes; TNF, tumor necrosis factor
Figure 5
Lamtor3 protein (red node) and its functional partners upon analysis by STITCH 5.0. Analysis of KEGG identified that Lamtor3 is directly involved in the MAPK signaling pathway, but may interface with other pathways including VEGF signaling and tumor formation such as those occurring in thyroid cancer, bladder cancer, endometrial cancer, glioma, non-small cell lung cancer, renal cell carcinoma, and melanoma through its interaction with Mapk1, Map2k1, Mapk3, Map2k2. Uniprot accession: Q5U204, Mass: 13,580 Da, Cellular location: Endosome. Lamtor3, Ragulator complex protein LAMTOR3; KEGG, Kyoto Encyclopedia of Genes and Genomes; VEGF, vascular endothelial growth factor
Figure 6
Map3k14 protein (red node) and its functional partners upon analysis by STITCH 5.0. Analysis of KEGG identified that Map3k14 is directly involved in NF-κB signaling, Epstein–Barr virus infection, TNF signaling, and MAPK signaling, and apoptosis. Uniprot accession: D3ZTD1, Mass: 103,584 Da, Cellular location: Cytosol and Nucleus. KEGG, Kyoto Encyclopedia of Genes and Genomes; Map3k14, Mitogen-activated protein kinase kinase kinase 14; TNF, tumor necrosis factor
Figure 7
Mapk8ip3 protein (red node) and its functional partners upon analysis by STITCH 5.0. Analysis of KEGG identified that Mapk8ip3 is directly involved in the MAPK signaling pathway. Mapk8ip3 may interface with activities that involve the JNK cascade because of its interaction with Mapk8, Mapk10, and Mapk9, which play important roles in apoptosis, inflammation, cytokine production, and metabolism activated in response to environmental stress. Uniprot accession: B0VXR4, Mass: 145,619 Da, Cellular location: Endoplasmic reticulum and Golgi apparatus. KEGG, Kyoto Encyclopedia of Genes and Genomes; JNK, c-Jun N-terminal kinase; Mapk8ip3, C-Jun-amino-terminal kinaseinteracting protein 3
Figure 8
Summarized hypothesized activities of AM and its related pathways. AM, α-mangostin; EMT, epithelial–mesenchymal transition; ERK, extracellular signal–regulated kinase; FAK, focal adhesion kinase; c-FLIP, Cflar also known as cellular FADD-like IL-1β-converting enzyme (FLICE)-inhibitory protein; IkB, inhibitors of NF-κB; IKKα, JNK, Mapk8ip3 also known as C-Jun N-terminal kinase; JIP3, JNK-interacting protein 3; JSAP, JNK-stress-activated protein kinase-associated protein 1; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MP1, Lamtor3 also known as MEK partner 1; NF-κB, nuclear factor κ-light-chain-enhancer of activated B cells; NIK, Map3k14 also known as NF-κB-inducing kinase; Raf, Rapidly accelerated fibrosarcoma proto-oncogene serine/threonine-protein kinase; Ras, rat sarcoma small GTPase; TGF-β, transforming growth factor-β; TNFR, Tumor necrosis factor receptor