Journal: PLoS ONE

Article Title: Hepatitis C Virus NS3/4A Protease Inhibits Complement Activation by Cleaving Complement Component 4

doi: 10.1371/journal.pone.0082094

Figure Lengend Snippet: (A) HCV NS3/4A protease, core, or NS5 was added to C4, and the products were separated by SDS-PAGE and subjected to CBB staining. Two approximately 17-kDa proteins (Fragment F1 and F2) and a 15-kDa protein (Fragment F3) were detected after incubation of C4 with HCV NS3/4A protease, but not after incubation with core or NS5. (B) Amino acid sequence of aa 1451-1620 region of C4. Protein fragments were analyzed by N-terminal peptide sequencing. The sequences of the N-termini of the 17-kDa proteins (Fragment F1 and F2) were SAEVCQCA and AEGKCPRQ, which are located at aa 1584–1591 and 1591–1598 in C4, respectively. The sequence of the N-terminus of the 15-kDa protein (Fragment F3) was EAPKVVEE, which is located at aa 1454–1461 in C4. (C) Schematic representation of C4γ chain, and Fragment F1, F2 and F3.

Article Snippet: Goat anti-human complement C4 antibody (MP Biomedicals, Santa Ana, CA) was bound to protein G–agarose beads (Thermo Scientific, Rockford, IL) in binding buffer (0.5% Nonidet P-40, 25 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA and protease inhibitor cocktail [Roche, Basel, Switzerland]) for 1 h at room temperature.

Techniques: SDS Page, Staining, Incubation, Sequencing

Journal: PLoS ONE

Article Title: Hepatitis C Virus NS3/4A Protease Inhibits Complement Activation by Cleaving Complement Component 4

doi: 10.1371/journal.pone.0082094

Figure Lengend Snippet: C4 was incubated in the presence or absence of HCV NS3/4A protease, and then C1-sensitized EA (EAC1) was added (EAC1-C4). After washing, C2 was added to form EAC1-C4-C2, and the complex was resuspended in C4d-GPS. The absorbance of the centrifuged supernatant was determined at 415 nm. The grade of hemolysis decreased in the presence of NS3/4A protease in a dose-dependent manner. All measurements were performed in triplicate, and data are expressed as means ± SD.

Article Snippet: Goat anti-human complement C4 antibody (MP Biomedicals, Santa Ana, CA) was bound to protein G–agarose beads (Thermo Scientific, Rockford, IL) in binding buffer (0.5% Nonidet P-40, 25 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA and protease inhibitor cocktail [Roche, Basel, Switzerland]) for 1 h at room temperature.

Techniques: Incubation

Journal: PLoS ONE

Article Title: Hepatitis C Virus NS3/4A Protease Inhibits Complement Activation by Cleaving Complement Component 4

doi: 10.1371/journal.pone.0082094

Figure Lengend Snippet: (A) VX950 was added to HCV NS3/4A protease at the indicated concentrations, and then C4 was added. Proteins were separated by SDS-PAGE for CBB staining. The three C4-derived fragments of 17 kDa and 15 kDa produced by NS3/4A protease action could not be detected after pretreatment with VX950, and this change was accompanied by an increased concentration of the 32-kDa C4γ chain. (B) The C4γ, 17-kDa, and 15-kDa bands were quantified by densitometric analysis using the Image J software. (C) C4 was incubated in the presence or absence of HCV NS3/4A or VX950, and then C1-sensitized EA (EAC1) was added (EAC1–C4). C2 and C4d-GPS were then added, and the absorbance of the supernatant was determined at 415 nm. Hemolysis was inhibited by NS3/4A protease and this inhibition was blocked by VX950. All measurements were made in triplicate, and data are expressed as means ± SD.

Article Snippet: Goat anti-human complement C4 antibody (MP Biomedicals, Santa Ana, CA) was bound to protein G–agarose beads (Thermo Scientific, Rockford, IL) in binding buffer (0.5% Nonidet P-40, 25 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA and protease inhibitor cocktail [Roche, Basel, Switzerland]) for 1 h at room temperature.

Techniques: SDS Page, Staining, Derivative Assay, Produced, Concentration Assay, Software, Incubation, Inhibition

Journal: PLoS ONE

Article Title: Hepatitis C Virus NS3/4A Protease Inhibits Complement Activation by Cleaving Complement Component 4

doi: 10.1371/journal.pone.0082094

Figure Lengend Snippet: (A) 293T cells were transfected with the indicated plasmids. Anti-C4 immunoprecipitates (IP) of supernatants were separated by SDS-PAGE and analyzed by immunoblotting with anti-C4γ antibody. Detergent-soluble cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-HA and anti-GAPDH antibodies. (B) 293T cells were transfected with the indicated plasmids. Culture supernatants were analyzed by immunoblotting with anti-C4γ antibody. Anti-C4 immunoprecipitates (IP) of supernatants were analyzed by immunoblotting with anti-C4 antibody. Detergent-soluble cell lysates were analyzed by immunoblotting with anti-HA and anti-GAPDH antibodies. (C) Huh7.5.1 cells were mock-infected or infected with HCVcc at a multiplicity of infection of 2 for 6 h, followed by mock-transfection or transfection with C4 expression plasmid. Culture supernatants and cell lysates were analyzed as described in (A) and (B). The anti-C4γ antibody was not appropriate for immunoblotting of IP samples derived from Huh7.5.1 cultures because of unavoidable nonspecific cross-reaction. * indicates non-specific reactions in (A) – (C).

Article Snippet: Goat anti-human complement C4 antibody (MP Biomedicals, Santa Ana, CA) was bound to protein G–agarose beads (Thermo Scientific, Rockford, IL) in binding buffer (0.5% Nonidet P-40, 25 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA and protease inhibitor cocktail [Roche, Basel, Switzerland]) for 1 h at room temperature.

Techniques: Transfection, SDS Page, Western Blot, Infection, Expressing, Plasmid Preparation, Derivative Assay

Journal: Frontiers in Oncology

Article Title: Establishment, characterization, and drug screening of low-passage patient individual non-small cell lung cancer in vitro models including the rare pleomorphic subentity

doi: 10.3389/fonc.2023.1089681

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: LYPD3 , APC , Sino Biological Europe GmbH, Düsseldorf, Germany , 11836-H08H.

Techniques: Cytometry

Journal: Frontiers in Oncology

Article Title: Establishment, characterization, and drug screening of low-passage patient individual non-small cell lung cancer in vitro models including the rare pleomorphic subentity

doi: 10.3389/fonc.2023.1089681

Figure Lengend Snippet: Flow cytometry. Histogram overlays of unstained controls (dotted lines for all three cell lines) and measurements for HROLu55 (green), HROLu22 (blue), and HROBML01 (red) for the epitopes CD326, PD-L1, EGFR, CD26, LYPD3, DSG3, CCD59, CD27, and CD90 are shown.

Article Snippet: LYPD3 , APC , Sino Biological Europe GmbH, Düsseldorf, Germany , 11836-H08H.

Techniques: Flow Cytometry

Journal: Disease Markers

Article Title: EV-Associated MMP9 in High-Grade Serous Ovarian Cancer Is Preferentially Localized to Annexin V-Binding EVs

doi: 10.1155/2017/9653194

Figure Lengend Snippet: EV isolation from ascites. EVs were isolated from ascites with the ligands AV (lane 1), CTB (lane 2), and STB (lane 3) and analyzed by western blotting. As a negative control, no ligand was added (lane 4). 250 μ l of pooled ascites from 6 (a) or 16 (b) ovarian cancer patients were used as starting material. The tetraspanin proteins CD9 and CD63, the transferrin receptor (CD71), and the cytosolic protein Alix were examined, and β -actin was used as a loading control. The normalized band intensities of the analyzed proteins are given below each band. The molecular weight markers are indicated on the left side of the images in kDa.

Article Snippet: The primary antibodies were mouse antibodies against the human proteins β -actin (C4), β -catenin (E-5), CD71 (H68.4), CD9 (C-4), Alix (1A12), CD59 (H-7), EpCAM (0.N.276) (Santa Cruz Biotechnology Inc., USA), CD63 (H5C6) (BD Biosciences, USA), and FN1-EDA (IST-9) (abcam PLC, UK).

Techniques: Isolation, Western Blot, Negative Control, Control, Molecular Weight

Journal: Disease Markers

Article Title: EV-Associated MMP9 in High-Grade Serous Ovarian Cancer Is Preferentially Localized to Annexin V-Binding EVs

doi: 10.1155/2017/9653194

Figure Lengend Snippet: Cancer proteins in EV isolations. (a) EVs isolated from 250 μ l of pooled ascites from 6 ovarian cancer patients (lanes 1–4) or from 5 patients with cirrhosis (lanes 5–8) were analyzed by western blot. As before the ligands AV, CTB, and STB were used for isolation, no ligand was added as a negative control. The protein cellular fibronectin (FN1-EDA), CD59, β -catenin, and epithelial cell adhesion molecule (EpCAM) were examined, and β -actin was used as loading control. The normalized band intensities of the analyzed proteins are given below each band. (b) EVs isolated from 500 μ l of the same samples as described for (a) were analyzed by zymography. The left part of the image (lanes 1–5) shows the ovarian cancer samples and the right part the cirrhosis samples (lanes 6–10). In lanes 1 and 6, 1 μ l of the pooled ascites samples were analyzed. The molecular weight markers are indicated on the left side of the images in kDa.

Article Snippet: The primary antibodies were mouse antibodies against the human proteins β -actin (C4), β -catenin (E-5), CD71 (H68.4), CD9 (C-4), Alix (1A12), CD59 (H-7), EpCAM (0.N.276) (Santa Cruz Biotechnology Inc., USA), CD63 (H5C6) (BD Biosciences, USA), and FN1-EDA (IST-9) (abcam PLC, UK).

Techniques: Isolation, Western Blot, Negative Control, Control, Zymography, Molecular Weight

Journal: Arthritis Research & Therapy

Article Title: Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

doi: 10.1186/ar4597

Figure Lengend Snippet: Confirmation of the nucleus pulposus cell phenotype in vitro. (A) Representative phase-contrast images of primary nucleus pulposus (NP) and annular fibrosus (AF) cells from donor 1 (D1) and donor 2 (D2). Passage numbers (P x ) for each donor’s tissue are indicated in parentheses (for example, D1 (P0)). Black bars = 20 μm. (B) Top : Graphed results of gene expression analysis of the chondrocyte markers collagen type I, α1 ( COL1A1 ), and collagen type II, α1 ( COL2A1 ), in primary AF (white bars) and NP (black bars) cell isolates from D1 (P0) and D2 (P1) tissues, respectively. Gene expression was normalized to β-actin (bACT) mRNA levels. Data presented are relative to the AF values. Lower panels : Immunoblot analysis of primary AF and NP cell lysates from two independent donors, D1 (P0) and D3 (P1), respectively, for COL1A1 and COL2A1. bACT was used as a loading control. (C) Representative phase-contrast images of AF and NP cultures from D4 and D5, both at P5. Black bars = 20 μm. (D) Immunoblot analysis of COL1A1, COL2A1 and SRY-box 9 (SOX9) on cell lysates from P5 AF and NP cells. (E) P5 AF and NP cells were stimulated for 7 days with differentiation medium, and cell lysates were analysed for COL1A1, COL2A1 and SOX9. bACT was used as a loading control. (F) Gene expression (mRNA) analysis of six NP markers: keratin 19 ( KRT19 ), Carbonic anhydrase XII ( CA12 ), cluster of differentiation 24 ( CD24 ), Forkhead box F1 ( FoxF1 ), paired box 1 ( Pax1 ) and pleiotrophin ( PTN ) in cultured AF (white bars) and NP cell isolates (black bars) in tissue from three independent donors: D2 (P1), D4 (P5) and D5 (P5). Marker gene expression was normalized to bACT levels. NP data presented are relative to AF values (per patient). a Indicated P -values were obtained by comparing AF and NP values combined for all three donors. Statistical significance was assessed by Student’s t -test. * P < 0.05; ** P < 0.01. P -values for AF vs NP comparisons per donor are listed in Additional file : Table S1.

Article Snippet: Antisera used were polyclonal goat anti-COL2A1 (1320-01; Southern Biotech, Birmingham, AL, USA), polyclonal goat collagen type I, α1 (anti-COL1A1) (1310-01; SouthernBiotech), polyclonal goat anti-SOX9 (ab3697; Abcam, Cambridge, UK), rabbit polyclonal antibody against brachyury T (hereafter ‘T’) (H-210 and SC-20109; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-SV40LTag (Pab 108, sc-148; Santa Cruz Biotechnology), rabbit polyclonal anti-TERT (R1187; Acris Antibodies, San Diego, CA, USA), mouse monoclonal β-actin (clone C4, 08691001; MP Biomedicals, Santa Ana, CA, USA), mouse monoclonal α-tubulin (clone B-5-1-2, T6074; Sigma-Aldrich), rabbit polyclonal KAP-1 (A300-275A; Bethyl Laboratories, Montgomery, TX, USA).

Techniques: In Vitro, Expressing, Western Blot, Cell Culture, Marker

Journal: Arthritis Research & Therapy

Article Title: Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

doi: 10.1186/ar4597

Figure Lengend Snippet: Marker expression in clonal subtypes. (A) Immunoblot analyses of collagen type II, α1 (COL2A1), SOX9 and collagen type II, α1 (COL1A1), in responder nucleus pulposus (NP-R) clones (left panels) and nonresponder nucleus pulposus (NP-nR) clones (right panels). Cell clones were incubated for 7 days with differentiation medium (Dmed; ‘D’ in figure) or maintenance medium (Mmed; ‘M’ in figure). The different subtype samples were run on the same gels to enable direct quantitative comparison. Three representative clones per NP subtype are shown. β-actin (bACT) was used as a loading control. Representative phase-contrast images of NP-R clones 108, 114 and 115 and of NP-nR clones 105, 113 and 119 are shown in the bottom row. Black bars = 20 μm. (B) (top right panel; continued in lower panels) Scatterplots of relative gene expression analysis of keratin 19 KRT19 , Carbonic anhydrase XII ( CA12 ), cluster of differentiation 24 ( CD24 ), Forkhead box F1 ( FOXF1 ), paired box 1 ( PAX1 ), pleiotrophin ( PTN ) and cartilage oligomeric matrix protein ( COMP ) in six representative NP-R clones (104, 108, 114, 115, 121 and 124; grey symbols) and six representative NP-nR clones (102, 105, 110, 113, 116 and 119; black symbols) at baseline ( t 0) (Mmed) or at 7 days of culture in Dmed. Gene expression was normalized to bACT mRNA levels. Log 2 -scaled expression data are presented relative to the average expression level of NP-nR at t 0. Each data point represents a triplicate measurement for an individual clone. The P -values were obtained by comparing the indicated experimental groups. Statistical significance was assessed by Student’s t -test. * P < 0.05; ** P < 0.01. Full list of P -values is available in Additional file : Table S2.

Article Snippet: Antisera used were polyclonal goat anti-COL2A1 (1320-01; Southern Biotech, Birmingham, AL, USA), polyclonal goat collagen type I, α1 (anti-COL1A1) (1310-01; SouthernBiotech), polyclonal goat anti-SOX9 (ab3697; Abcam, Cambridge, UK), rabbit polyclonal antibody against brachyury T (hereafter ‘T’) (H-210 and SC-20109; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-SV40LTag (Pab 108, sc-148; Santa Cruz Biotechnology), rabbit polyclonal anti-TERT (R1187; Acris Antibodies, San Diego, CA, USA), mouse monoclonal β-actin (clone C4, 08691001; MP Biomedicals, Santa Ana, CA, USA), mouse monoclonal α-tubulin (clone B-5-1-2, T6074; Sigma-Aldrich), rabbit polyclonal KAP-1 (A300-275A; Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Marker, Expressing, Western Blot, Clone Assay, Incubation

Journal: Arthritis Research & Therapy

Article Title: Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

doi: 10.1186/ar4597

Figure Lengend Snippet: Extracellular matrix molecules in nucleus pulposus differentiation. (A) Extracellular matrix (ECM) properties in responsive nucleus pulposus (NP-R) clones (left column; R) and nonresponsive nucleus pulposus (NP-nR) clones (right columns; nR). Sulphated glucosaminoglycan (GAG; left graph) and hydroxyproline (right graph; OH-pro) quantities in four representative NP-R clones (108, 114, 115 and 124; grey circles) and four NP-nR clones (102, 105, 113 and 119; black circles) at baseline ( t = 0), at 7 days in differentiation medium (Dmed) and at 7 days in maintenance medium (Mmed). Statistical significance was assessed by Student’s t -test. * P < 0.05; ** P < 0.01; n.dct ., Not detectable. Full list of P -values is provided in Additional file : Table S3. (B) Comparison of marker expression on monolayer (mono; uncoated polystyrene), Matrigel-coated (M.gel) and aggrecan- coated (ACAN) cultured NP cell clones. Gene expression levels of SRY-box 9 ( SOX9 ), COL2A1 , ACAN and cartilage oligomeric matrix protein ( COMP ) are depicted. Grey bars correspond to NP-R115, and black bars to clone NP-nR105. Gene expression was normalized to β-ACTIN mRNA levels. Data are expressed as fold changes relative to expression levels at baseline ( t = 0) (Mmed). Statistical significance was assessed by Student’s t -test. Full list of P -values is provided in Additional file : Table S4. * P < 0.05; ** P < 0.01; n.dct. = not detectable. (C) Phase-contrast images of representative NP-R clones and NP-nR clones on Matrigel-coated plates at 36 hours postplating. (D) Phase-contrast images of representative NP-R clones and NP-nR clones on monolayer plastic or ACAN coating at 72 hours postplating. Black bars = 20 μm.

Article Snippet: Antisera used were polyclonal goat anti-COL2A1 (1320-01; Southern Biotech, Birmingham, AL, USA), polyclonal goat collagen type I, α1 (anti-COL1A1) (1310-01; SouthernBiotech), polyclonal goat anti-SOX9 (ab3697; Abcam, Cambridge, UK), rabbit polyclonal antibody against brachyury T (hereafter ‘T’) (H-210 and SC-20109; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-SV40LTag (Pab 108, sc-148; Santa Cruz Biotechnology), rabbit polyclonal anti-TERT (R1187; Acris Antibodies, San Diego, CA, USA), mouse monoclonal β-actin (clone C4, 08691001; MP Biomedicals, Santa Ana, CA, USA), mouse monoclonal α-tubulin (clone B-5-1-2, T6074; Sigma-Aldrich), rabbit polyclonal KAP-1 (A300-275A; Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Clone Assay, Marker, Expressing, Cell Culture