Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: HPSE1 knockdown and upregulation efficiency in OSCC cells. The efficiency of endogenous HPSE1 modulation was verified by Immunofluorescence (A) , Western Blot (B) and RT-qPCR (C) analyzes. For the loss-of-function strategy, SCC-9 cells were transduced with shRNA expressing a vector sequence against HPSE1 (shRNA HPSE1−) and empty vector (scrRNA control). For the gain-of-function strategy, SCC-9 cells were transduced with a ORF clone in a shuttle vector to enhance HPSE1 expression (orfRNA HPSE1+), along with mock-transduced cells as described in Methods . shRNA HPSE1− cells showed a significant reduction and orfRNA HPSE+ cells showed a significant upregulation of HPSE1 in both mRNA and protein levels compared to scrRNA control and parental mock-transduced SCC-9 control cells (pSCC9), without targeting sequences. Results were statistically determined by ANOVA, followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Knockdown, Immunofluorescence, Western Blot, Quantitative RT-PCR, Transduction, shRNA, Expressing, Plasmid Preparation, Sequencing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Downregulation of HPSE1 inhibits proliferation and enhances apoptosis of OSCC cells. Cells were subjected to MTT cell proliferation (A) , DNA content cell cycle analysis (B) , and apoptosis (C) assays. (A) Cell Proliferation Assay of the OSCC SCC9 cell line (Control) compared to empty vector SCC9 control clone (ScrRNA control), HPSE1 inhibitory/silencing SCC9-ShRNA HPSE1− clone, and the HPSE1 overexpressing SCC9-OrfRNA HPSE1+ clone. (B) Quantification of cell cycle analysis was performed by flow cytometry after staining with propidium iodide for the SCC-9 control cells and the respectives HPSE1-modulated clones. Abrogation of HPSE1 induced arrest of the SCC-9 cells cycle in G1 phase, and its upregulation significantly induced cell proliferation. (C) Flow cytometric analysis of apoptosis showed a remarkable increase in the number of apoptotic cells in HPSE1-silenced cells (ShRNA HPSE1−), while SCC-9 overexpressing HPSE1 (Orf-RNA HPSE1+) exhibited a reduction in apoptosis. (D) No significant differences in cell adhesion properties were observed among any transduced clones modulating HPSE1 expression, compared to parental mock-transduced cells (control). Plots compose experimental triplicate analysis and were statistically calculated using ANOVA followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: MTT Cell Proliferation, Cell Cycle Assay, Proliferation Assay, Control, Plasmid Preparation, shRNA, Flow Cytometry, Staining, Clone Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Overexpression of HPSE1 is associated with migration, invasion, ECM remodeling, and acquisition of EMT properties. (A) Photomicrographs of cell lines were taken 0, 24, and 48 h after wounding (40X). The average width of the lacunae was measured. Migration of SCC-9 cells was significantly decreased in HPSE1-silenced cells (ShRNA HPSE1−) and increased in HPSE1-upregulated clones (OrfRNA HPSE1+). Migration analysis based on this assay showed that cells with lower expression of HPSE1 closed the scratch wound significantly slower than the SCC9 Control cells. (B) Invasion of SCC-9 cells was significantly inhibited by HPSE1 knockdown, and significantly enhanced after its upregulation. (C) Analysing EMT markers, the downregulation of HPSE1 significantly induced the expression of E-Cadherin (E-CAD), while OrfRNA HPSE1+ SCC9 clones overexpressing heparanase had a significant increasing of Vimentin (VIM) and SNAIL expressions. (D) The upregulation of HPSE1 significantly enhanced the expressions of MMP2 and MMP9. All the graphs compile experimental triplicate analyses, and the results were obtained by ANOVA followed by Tukey assay, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Migration, shRNA, Clone Assay, Expressing, Control, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Overexpression of HPSE1 enhances tumor neo-vascularization and induces VEGFA expression. HUVEC cells on a Miogel 2D layer co-culture. Photomicrograph of HUVEC endothelial cells added with preconditioned medium from OSCC cell lines and their respective clones after 12 h of experiment: (A) HUVEC cells incubated with preconditioned medium of SCC9-OrfRNA HPSE1+ clone overexpressing HPSE1 (100×); (B) HUVEC cells incubated with preconditioned medium of parental SCC9 (Control) (100×); (C) HUVEC cells incubated with preconditioned medium of SCC9-ShRNA HPSE1- clone with reduction of HPSE1 expression (100×). (D) Quantification of measures of the vessel circumference perimeter for each one of the conditions; the graph compiles two experimental triplicate analysis. (E) Gene expression analysis of endothelial growth factor VEGFA by qRT-PCR; the graph compiles folded expression values of relative quantification by ddCT obtained through comparison of clones OrfRNA HPSE1+ and ShRNA HPSE1− relative to the control SCC9 cells (normal reference = 1). Normalization of the analysis was performed using the endogenous control gene, PPIA. Statistical tests were performed using ANOVA followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Expressing, Co-Culture Assay, Clone Assay, Incubation, Control, shRNA, Gene Expression, Quantitative RT-PCR, Quantitative Proteomics, Comparison

Journal: Oncotarget

Article Title: Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-β autocrine loop

doi: 10.18632/oncotarget.11450

Figure Lengend Snippet: Human liver hepatocellular cells (HepG2) were stimulated in the presence or absence of 2 μM S1P for 72 h. ( A ) S1P increased mRNA expression of S1P 1 . HepG2 cells were treated with S1P 1 inhibitor W146 (10 μM), HPSE inhibitor low molecular weight heparin (50 μg/ml), generic MMP inhibitor Ilomastat (GM6601, 10 μM), or GM6001 negative control (NC) for 30 min before S1P stimulation for 72 h. ( B ) S1P downregulated mRNA transcripts of syndecan-1, which was blocked by W146 and heparin, but was not by Ilomastat. ( C ) S1P-induced sGAG release from HepG2 cells was eliminated by W146, heparin, and Ilomastat, respectively. sGAG in the culture medium was detected by using 1,9-dimethylmethylene blue (DMMB) assay. ( D ) S1P decreased syndecan-1 staining ( green ) but induced translocation of syndecan-1 into the nucleus (DAPI, blue ). Confocal images depict a z -plane through the center of the cell nucleus. Scale bar, 10 μm. ( E ) and ( F ), a representative image of Western blots (E) with densitometric quantification (F) of syndecan-1 in cell lysates after 30 min exposure of W146, heparin, and Ilomastat before S1P treatment for 72 h. Results are presented as a ratio to GAPDH. ( G ) and ( H ), immunofluorescence images (maximum-intensity projections of a confocal z -stack) (G) and the coverage of heparan sulfate (HS, green ). Nuclei were counterstained with DAPI ( blue ) (H). * P < 0.05 as compared with CT; # P < 0.05 as compared with S1P (ANOVA).

Article Snippet: Noneffective scrambled (TR30021) -, HPSE (TL307138) -, MMP-7 (TL311438) -, syndecan-1 (TL309598) -shRNA plasmid (OriGene, USA) respective targeting human HPSE (NM_006665), MMP-7 (NM_002423), and syndecan-1 (NM_002997) were used to obtain the negative control (NC), HPSE-, MMP-7-, syndecan-1-stable silenced HepG2 cell lines according to the manufacturer's instructions.

Techniques: Expressing, Molecular Weight, Negative Control, Dimethylmethylene Blue Assay, Staining, Translocation Assay, Western Blot, Immunofluorescence

Journal: Oncotarget

Article Title: Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-β autocrine loop

doi: 10.18632/oncotarget.11450

Figure Lengend Snippet: HPSE knockdown (HPSE shRNA) and MMP-7 knockdown (MMP-7 shRNA) by shRNA transduction were performed. Then, cells were treated with S1P 1 inhibitor W146 (10 μM), and HPSE inhibitor low molecular weight heparin (50 μg/ml) for 30 min before S1P stimulation for 72 h. Cells transfected with scrambled shRNA were set as a negative control (NC). HPSE expression and activity were modulated by S1P via S1P 1 . ( A ) HPSE mRNA expression levels in WT and HPSE shRNA cells were determined by qRT-PCR. The data were corrected to GAPDH as an internal control and represents the mean ± S.D. (Error bars) of at least three independent experiments performed in triplicate, and final results were normalized to the non-treated WT control cells. ( B ) and ( C ), HPSE activity in protein extracts (B) and conditioned media (C) in WT and HPSE shRNA cells. HPSE activity is expressed as HS removed rate, nanograms per minute. ( D ) MMPs activity in the culture medium was determined by using Sensolyte 390 generic MMP activity kit (AnaSpec). The results were normalized to the WT control cells. ( E ) MMP-7 mRNA expression levels in WT, HPSE shRNA, and MMP-7 shRNA cells. ( F ) MMP-7 activity in the culture medium was determined by using SensoLyte 520 MMP-7 assay kit (AnaSpec). MMP-7 shRNA and WT control cells were treated with 2 μM S1P for 72 h. Then, sGAG assay, syndecan-1 expression, and staining of HS were performed. ( G ) sGAG in the culture medium was measured by DMMB assay. ( H ) a representative image of Western blots with densitometric quantification of syndecan-1. ( I ), the coverage of heparan sulfate (HS) was estimated by using maximum-intensity projections of a confocal z -stack. * P < 0.05, ** P < 0.01 as compared with CT; # P < 0.05 as compared with S1P-treated WT control (ANOVA).

Article Snippet: Noneffective scrambled (TR30021) -, HPSE (TL307138) -, MMP-7 (TL311438) -, syndecan-1 (TL309598) -shRNA plasmid (OriGene, USA) respective targeting human HPSE (NM_006665), MMP-7 (NM_002423), and syndecan-1 (NM_002997) were used to obtain the negative control (NC), HPSE-, MMP-7-, syndecan-1-stable silenced HepG2 cell lines according to the manufacturer's instructions.

Techniques: Knockdown, shRNA, Transduction, Molecular Weight, Transfection, Negative Control, Expressing, Activity Assay, Quantitative RT-PCR, Control, Staining, Dimethylmethylene Blue Assay, Western Blot

Journal: Oncotarget

Article Title: Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-β autocrine loop

doi: 10.18632/oncotarget.11450

Figure Lengend Snippet: HepG2 WT and HPSE shRNA cells were stimulated with 2 μM S1P for 72 h with or without pretreatment for 30 min with S1P 1 inhibitor W146 (10 μM), PI3K/AKT inhibitor LY294002 (20 μM) or ERK1/2 inhibitor GDC-0994 (50 μM). ( A ) and ( B ), the phosphorylation levels of AKT (A) and ERK1/2 (B) in WT and HPSE shRNA cells were evaluated by Western blot. Histograms show densitometric quantification of p -AKT or p -ERK normalized to total AKT or ERK, respectively. ( C ) LY294002 inhibited the S1P-induced expression of HPSE mRNA, but GDC-0994 did not. ( D ) and ( E ) LY294002 abolished the S1P-induced mRNA expression and activity of MMP-7, but GDC-0994 did not. The mRNA expression of MMP-7 in cell lysates was performed by qRT-PCR, and the activity of MMP-7 in cell mediums was determined by using SensoLyte 520 MMP-7 assay kit (AnaSpec). ( F ) S1P-induced sGAG release was blocked by LY294002 or in partly inhibited by GDC-0994. sGAG in culture medium was detected by DMMB assay. ( G ) and ( H ), effects of S1P in the suppression of syndecan-1 expression (G) and decrease in the coverage of HS (H) were blocked by LY294002 or in partly inhibited by GDC-0994. Syndecan-1 expression was measured by Western blot. The coverage of heparan sulfate (HS) was estimated by using maximum-intensity projections of a confocal z -stack. * P < 0.05 as compared with WT control cells; # P < 0.05 as compared with S1P-treated WT control (ANOVA).

Article Snippet: Noneffective scrambled (TR30021) -, HPSE (TL307138) -, MMP-7 (TL311438) -, syndecan-1 (TL309598) -shRNA plasmid (OriGene, USA) respective targeting human HPSE (NM_006665), MMP-7 (NM_002423), and syndecan-1 (NM_002997) were used to obtain the negative control (NC), HPSE-, MMP-7-, syndecan-1-stable silenced HepG2 cell lines according to the manufacturer's instructions.

Techniques: shRNA, Phospho-proteomics, Western Blot, Expressing, Activity Assay, Quantitative RT-PCR, Dimethylmethylene Blue Assay, Control

Journal: Oncotarget

Article Title: Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-β autocrine loop

doi: 10.18632/oncotarget.11450

Figure Lengend Snippet: HepG2 WT, HPSE shRNA, and syndecan-1 shRNA cells were treated with 2 μM S1P for 72 h with or without pretreatment for 30 min with S1P 1 inhibitor W146 (10 μM), ERK1/2 inhibitor GDC-0994 (50 μM), or 5 μg/ml neutralizing TGF-β1 antibody as indicated. ( A ), ( B ), and ( C ), S1P promoted TGF-β1 secretion in culture medium (A), and mRNA (B) and protein expression (C) as measured by ELISA, qRT-PCR and Western blot assay, respectively. Representative image of Western blots with densitometric quantification are shown for TGF-β1, and the results represent the mean ± S.D. as a ratio to GAPDH. ( D ) the knockdown of syndecan-1 by shRNA transduction was checked. HepG2 cells were stimulated with 0.5 ng/ml exogenous TGF-β1 for 72 h. ( E ) change in expression of HPSE mRNA in cells treated with TGF-β1 was not detected. HepG2 cells were pretreated with ERK1/2 inhibitor GDC-0994 (50 μM) for 30 min before exposure to 0.5 ng/ml exogenous TGF-β1 for 72 h. ( F ) TGF-β1 not significantly changed syndecan-1 mRNA expression. HepG2 WT and MMP-7 shRNA cells were stimulated with 0.5 ng/ml exogenous TGF-β1 for 72 h. ( G ) knockdown of MMP-7 abolished the TGF-β1-induced sGAG release. * P < 0.05, ** P < 0.01 as compared with WT control cells; # P < 0.05 as compared with S1P-treated WT control (ANOVA).

Article Snippet: Noneffective scrambled (TR30021) -, HPSE (TL307138) -, MMP-7 (TL311438) -, syndecan-1 (TL309598) -shRNA plasmid (OriGene, USA) respective targeting human HPSE (NM_006665), MMP-7 (NM_002423), and syndecan-1 (NM_002997) were used to obtain the negative control (NC), HPSE-, MMP-7-, syndecan-1-stable silenced HepG2 cell lines according to the manufacturer's instructions.

Techniques: shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Knockdown, Transduction, Control

Journal: Oncotarget

Article Title: Sphingosine-1-phosphate induced epithelial-mesenchymal transition of hepatocellular carcinoma via an MMP-7/syndecan-1/TGF-β autocrine loop

doi: 10.18632/oncotarget.11450

Figure Lengend Snippet: HepG2 control and syndecan-1 shRNA cells were treated with 2 μM S1P for 72 h with or without an addition of 5 μg/ml neutralizing TGF-β1 antibody. ( A ) and ( B ), HepG2 cells knocked down for syndecan-1 (syndecan-1 shRNA) compared with control cells were plated to invade through Matrigel-coated Transwell. An addition of 5 μg/ml neutralizing TGF-β1 antibody abolished S1P-induced cell invasion. The invaded cells were quantified and representative images are present. ( C ), ( D ), and ( E ), representative images were photographed under a scanning electron microscope (C). Alterations in cell area (D) and morphology (the aspect ratio and circularity, E) were evaluated by using image J. The results represent the mean ± S.D. of 80 cells from at least three images. ( F ) expression of epithelial phenotype marker E-cadherin and mesenchymal phenotype marker Vimentin, as well as transcription regulatory protein Snail, were analyzed by Western blotting with densitometric quantification. ( G ) a schematic summary of S1P-induced EMT via an MMP-7/syndecan-1/TGF-β1 autocrine loop. This pathway involves activation of PI3K/AKT signaling pathways via S1P 1 , which triggers an enzyme HPSE, leading to an increase in expression and activity of MMP-7 and leading to activation of the ERK1/2 signaling pathway. MMP-7 mediated the shedding of syndecan-1. ERK1/2 mediates the suppression of syndecan-1. The loss of syndecan-1 causes an increase in TGF-β1 secretion and expression. The limited protracted increase in TGF-β1 further enhances MMP-7 activity, leading to syndecan-1 shedding, thus forming an MMP-7/syndecan-1/TGF-β1 autocrine loop. Finally, TGF-β1 and syndecan-1 are essential for S1P-induced epithelial to mesenchymal transition. Syndecan-1 probably behaves as a brake of the autocrine loop and an operator modulated forming of the metastasis-permissive microenvironment. * P < 0.05 as compared with WT control cells (ANOVA).

Article Snippet: Noneffective scrambled (TR30021) -, HPSE (TL307138) -, MMP-7 (TL311438) -, syndecan-1 (TL309598) -shRNA plasmid (OriGene, USA) respective targeting human HPSE (NM_006665), MMP-7 (NM_002423), and syndecan-1 (NM_002997) were used to obtain the negative control (NC), HPSE-, MMP-7-, syndecan-1-stable silenced HepG2 cell lines according to the manufacturer's instructions.

Techniques: Control, shRNA, Microscopy, Expressing, Marker, Western Blot, Activation Assay, Protein-Protein interactions, Activity Assay

Journal: Thyroid Research

Article Title: A preliminary study developing a scoring model incorporating fibrinogen-like protein 2 for predicting glucocorticoid resistance in thyroid eye disease

doi: 10.1186/s13044-026-00293-8

Figure Lengend Snippet: Proteomics analysis comparing responders with non-responders in TED. ( A ) Volcano plot showing differential protein abundance between ivGC responders and non-responders. The y-axis represents -log10 unadjusted p-values. Multiple testing correction was performed using the Benjamini-Hochberg method; however, no proteins remained significant after FDR correction (q < 0.05). Therefore, unadjusted P values are shown for hypothesis-generating visualization. Candidate biomarkers are highlighted with circles. ( B ) PPI network analysis centered on TGF-β. Interactions were analyzed using the STRING database, illustrating the key regulatory nodes and connections among the DAPs. The network showed significant enrichment of protein-protein interactions (PPI enrichment p = 4.48 × 10 − 7 ). ARPC5, actin-related protein 2/3 complex subunit 5; CAH1, carbonic anhydrase 1; CAH2, carbonic anhydrase 2; CMGA, chromogranin A; DAPs, differentially abundant proteins; DIA, data-independent acquisition; FGL2, fibrinogen-like protein 2; PGN, progranulin; HPA, heparanase; INHBE, inhibin beta E chain; MBL2, mannose-binding lectin 2; MGAT1, alpha-1,3-mannosyl-glycoprotein; PPI, protein–protein interaction; PRDX6, peroxiredoxin 6; TED, thyroid eye disease; TGF-β, transforming growth factor beta; UROM, uromodulin; VTN, vitronectin

Article Snippet: Serum fibrinogen-like protein 2 (FGL2), transforming growth factor beta (TGF-β), and heparanase were measured using commercially available ELISA kits, as follows: FGL2 (LEGEND MAXTM Human sFGL2 ELISA kit, Cat. No. 381558, BioLegend, San Diego, California, USA; intra-assay coefficient of variation [CoV] 5.9%–8.4%, inter-assay CoV 8.7%–10.8%, standard range 0.25–16 ng/mL), TGF-β (Human TGF-β1 Sandwich ELISA kit, Cat. No. KE00002, Proteintech; intra-assay CoV 4.5%–4.9%, inter-assay CoV 2.0%–9.4%, standard range 15.6–1000 pg/mL), and heparanase (Human Heparanase ELISA kit, Cat. No. CSB-E09899h, CUSABIO; intra-assay CoV < 8%, inter-assay CoV < 10%, standard range 0.156–10 ng/mL).

Techniques: Quantitative Proteomics, Protein-Protein interactions, Data-independent acquisition, Binding Assay

Journal: Thyroid Research

Article Title: A preliminary study developing a scoring model incorporating fibrinogen-like protein 2 for predicting glucocorticoid resistance in thyroid eye disease

doi: 10.1186/s13044-026-00293-8

Figure Lengend Snippet: Comparison of protein abundance among the four groups in the DIA proteomics analysis of the 12 candidate biomarker proteins based on the criteria described in the Methods. TGF-β, FGL2, and heparanase, which also showed significant differences between responders and non-responders with TED in the four-group comparison, were selected as final candidate biomarkers. Statistical significance was assessed by the Kruskal–Wallis test. * P < 0.05; ** P < 0.01. DIA, data-independent acquisition; FGL2, fibrinogen-like protein 2; TED, thyroid eye disease; TGF-β, transforming growth factor beta

Article Snippet: Serum fibrinogen-like protein 2 (FGL2), transforming growth factor beta (TGF-β), and heparanase were measured using commercially available ELISA kits, as follows: FGL2 (LEGEND MAXTM Human sFGL2 ELISA kit, Cat. No. 381558, BioLegend, San Diego, California, USA; intra-assay coefficient of variation [CoV] 5.9%–8.4%, inter-assay CoV 8.7%–10.8%, standard range 0.25–16 ng/mL), TGF-β (Human TGF-β1 Sandwich ELISA kit, Cat. No. KE00002, Proteintech; intra-assay CoV 4.5%–4.9%, inter-assay CoV 2.0%–9.4%, standard range 15.6–1000 pg/mL), and heparanase (Human Heparanase ELISA kit, Cat. No. CSB-E09899h, CUSABIO; intra-assay CoV < 8%, inter-assay CoV < 10%, standard range 0.156–10 ng/mL).

Techniques: Comparison, Quantitative Proteomics, Biomarker Discovery, Data-independent acquisition

Journal: Archives of biochemistry and biophysics

Article Title: Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

doi: 10.1016/j.abb.2011.02.014

Figure Lengend Snippet: Fig. 1. Effect of simultaneous expression of FALS hSOD1 and hCCS on SOD1- containing HMWS in AAV293 cells. (A) Schematic representation of plasmid vectors used to produce dicistronic mRNA for hSOD1 and hCCS co-expression. B, BamHI; X, XhoI; N, NcoI; K, KpnI; RI, EcoRI. (B) Detection of mutant SOD1 with or without CCS revealed with reducing gel (see Materials and methods). Top panel shows a hSOD1 blot visualized by ECL chemiluminescence. Asterisks depict the high molecular- weight species. The CCS proteins were also detected by the sheep polyclonal hSOD1 (574597) antibody due to the high homology of the two proteins. Middle panel shows the identical SOD1 blot as the top panel, except with shorter exposure time. Lower panel represents a CCS blot. This figure shows typical results obtained with three independent experiments.

Article Snippet: The primary antibodies used were as follows: sheep polyclonal anti-human Cu,Zn SOD1 (574597) (1:2000; Calbiochem, La Jolla, CA, USA), rabbit polyclonal anti-human Cu,Zn SOD1 (RDI-SODabRx) (1:5000; Fitzgerald, Concord, MA, USA), mouse monoclonal anti-mitochondrial Hsp70 (MN3-028) (1:5000; Affinity Bioreagents, Golden, CO), mouse monoclonal anti-human CCS (2A1) (1:5000; Abcam, Cambridge, MA, USA), mouse monoclonal anti-actin (1:10,000; Sigma), and mouse monoclonal anti-Hsp70 (W27) (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Expressing, Plasmid Preparation, Mutagenesis, High Molecular Weight

Journal: Archives of biochemistry and biophysics

Article Title: Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

doi: 10.1016/j.abb.2011.02.014

Figure Lengend Snippet: Fig. 2. Co-expression of CCS reduced the non-ionic detergent-insoluble fraction of FALS A4V and G85R SOD1 mutants. As described in Materials and methods, ‘‘PBS- soluble fraction’’ (lane 1), ‘‘non-ionic detergent-soluble fraction’’ (lane 2), and ‘‘non- ionic detergent-insoluble fractions’’ (lane 3) were obtained and analyzed. Proteins (5 lg) were loaded onto 10–20% gradient gels. Blots were obtained with rabbit polyclonal antibody (RDI-SODabRx) to Cu,Zn SOD1, or with monoclonal antibody to CCS, and visualized by ECL chemiluminesence.

Article Snippet: The primary antibodies used were as follows: sheep polyclonal anti-human Cu,Zn SOD1 (574597) (1:2000; Calbiochem, La Jolla, CA, USA), rabbit polyclonal anti-human Cu,Zn SOD1 (RDI-SODabRx) (1:5000; Fitzgerald, Concord, MA, USA), mouse monoclonal anti-mitochondrial Hsp70 (MN3-028) (1:5000; Affinity Bioreagents, Golden, CO), mouse monoclonal anti-human CCS (2A1) (1:5000; Abcam, Cambridge, MA, USA), mouse monoclonal anti-actin (1:10,000; Sigma), and mouse monoclonal anti-Hsp70 (W27) (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Expressing

Journal: Archives of biochemistry and biophysics

Article Title: Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

doi: 10.1016/j.abb.2011.02.014

Figure Lengend Snippet: Fig. 5. Complexes formed between G85R and CCS, and between SOD1 and Hsp70 detected by co-immunoprecipitation. AAV293 cells were transiently transfected with (A and C) G85R-GFP or G85R-CCS vectors and with (B) FLAG-tagged WT SOD1 or A4V mutant expression vectors with or without CCS co-expression. At 48 h post- transfection, cells were lysed in lysis buffer (20 mM Hepes, 1% Triton X-100, 10% glycerol, pH 7.4) that contained protease inhibitor cocktail (Sigma). After brief sonication, cell lysates were centrifuged (10,000g for 5 min) and the supernatant was collected. Rabbit polyclonal antibody that does not cross-react with CCS, against SOD1 (RDI-SODabRx) (A) or mouse monoclonal antibody against Hsp70 (MN3-028) (B and C) were added to a supernatant aliquot containing 500 lg of cellular proteins and incubated for 1 h at 4 C. To isolate the SOD1 antibody conjugated proteins (A) or the Hsp70 antibody conjugated proteins (B and C), protein A-conjugated agarose beads (Pierce) were added and the mixture incubated for another 1 h at 4 C. After washing with wash buffer, the immunoprecipitates were recovered with 1 SDS sample buffer, separated by SDS–PAGE, and probed with rabbit anti-SOD1 (RDI-SODabRx), mouse monoclonal anti-CCS (2AI) and anti- Hsp70 antibodies (MN3-028) as indicated. IB, immunoblotting; IP, immunoprecipitation.

Article Snippet: The primary antibodies used were as follows: sheep polyclonal anti-human Cu,Zn SOD1 (574597) (1:2000; Calbiochem, La Jolla, CA, USA), rabbit polyclonal anti-human Cu,Zn SOD1 (RDI-SODabRx) (1:5000; Fitzgerald, Concord, MA, USA), mouse monoclonal anti-mitochondrial Hsp70 (MN3-028) (1:5000; Affinity Bioreagents, Golden, CO), mouse monoclonal anti-human CCS (2A1) (1:5000; Abcam, Cambridge, MA, USA), mouse monoclonal anti-actin (1:10,000; Sigma), and mouse monoclonal anti-Hsp70 (W27) (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Immunoprecipitation, Transfection, Mutagenesis, Expressing, Lysis, Protease Inhibitor, Sonication, Incubation, SDS Page, Western Blot