Journal: Cancers

Article Title: Clinical Significance of SERPINA1 Gene and Its Encoded Alpha1-Antitrypsin Protein in NSCLC

doi: 10.3390/cancers11091306

Figure Lengend Snippet: SERPINA1 expression is downregulated in NSCLC and is a prognostic factor for overall and disease-free survival. ( A ) Relative expression (ΔCt) of SERPINA1 in tumor and in paired non-tumor lung tissues. SERPINA1 expression was normalized to reference genes ESD and RPS18 . A higher relative value means a lower gene expression. ( B ) SERPINA1 expression ratio (tumor vs. non-tumor lung tissue) in 351 NSCLC patients. Dotted line indicates equal expression in tumor and lung tissue. ( C – F ). Kaplan-Meier overall and disease-free survival curves for all patients. ( G ) and ( H ) Kaplan-Meier overall and disease-free survival curves for current smokers. The program cut-off finder ( http://molpath.charite.de/cutoff/ ) was used to define the values separating the groups. p < 0.05 was considered significant.

Article Snippet: For overexpression of SERPINA1 , cells were transfected with the pCMV6-SERPINA1 (SC119951, OriGene, Rockville, MD, United States) vector or with the pCMV6 (PS100020, OriGene) vector as a control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Expressing, Gene Expression

Journal: Cancers

Article Title: Clinical Significance of SERPINA1 Gene and Its Encoded Alpha1-Antitrypsin Protein in NSCLC

doi: 10.3390/cancers11091306

Figure Lengend Snippet: Cox Regression Analyses.

Article Snippet: For overexpression of SERPINA1 , cells were transfected with the pCMV6-SERPINA1 (SC119951, OriGene, Rockville, MD, United States) vector or with the pCMV6 (PS100020, OriGene) vector as a control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Gene Expression

Journal: Cancers

Article Title: Clinical Significance of SERPINA1 Gene and Its Encoded Alpha1-Antitrypsin Protein in NSCLC

doi: 10.3390/cancers11091306

Figure Lengend Snippet: Exogenously added AAT induces cancer cell migration, increases viability and resistance against staurosporine-induced apoptosis. ( A ) SERPINA1 expression relative to housekeeping gene POLR2A in H1975 and H661 cell lines. Data presented as the mean ± SD from three independent measurements. ( B ) Confocal immunofluorescence staining images of AAT protein (green) in H1975 and H661 in control and AAT (2 mg/mL) treated cells for 18 hours in serum-free medium. Nuclei were defined by DAPI (blue). Scale bar: 30 µm. ( C ) and ( D ) Migratory properties of H1975 and H661 cells without and with pre-treatment of AAT (2 mg/mL). The migrated cells were stained with 0.1% crystal violet. The graphs indicate data from three independent experiments as mean ± SD. p < 0.05 was considered as significant. ( E ) H1975 and H661 cells were treated with 50 nM staurosporine (STS) alone or together with AAT (0.05–2 mg/mL) for 18 hours and lactate dehydrogenase (LDH) release was measured. Each point is a mean ± SD from six independent experiments. ( F ) H1975 and H661 cells were treated with 50 nM STS alone or together with AAT (0.05–2 mg/mL) for 18 hours. Annexin V surface expression was determined by flow cytometry. Experiments were repeated four times. Each point represents a mean ± SD. p < 0.05 was considered as significant.

Article Snippet: For overexpression of SERPINA1 , cells were transfected with the pCMV6-SERPINA1 (SC119951, OriGene, Rockville, MD, United States) vector or with the pCMV6 (PS100020, OriGene) vector as a control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Migration, Expressing, Immunofluorescence, Staining, Control, Flow Cytometry

Journal: Cancers

Article Title: Clinical Significance of SERPINA1 Gene and Its Encoded Alpha1-Antitrypsin Protein in NSCLC

doi: 10.3390/cancers11091306

Figure Lengend Snippet: Silencing or overexpressing SERPINA1 gene affects cancer cell colony formation, migration, viability and resistance to apoptosis. ( A ) and ( B ) Cancer colony number per area in SERPINA1 siRNA-treated H1975 cells or SERPINA1 overexpressing H661 cells. Each point represents a separate experiment. Data presented as mean ± SD. p < 0.05 was considered as significant. ( C ) and ( D ) The migratory abilities of H1975 cells after SERPINA1 -knockdown and H661 cells after SERPINA1 overexpression with/without addition of AAT. The graphs indicate data from three independent experiments as mean ± SD. p < 0.05 was considered as significant. ( E ) LDH release rate after silencing or overexpression of SERPINA1 . H1975 and H661 cells were treated with 50 nM STS alone or together with AAT (2 mg/mL) for 18 h after SERPINA1 knockout or overexpression, respectively. All experiments were performed in duplicates and repeated three times. Each point represents a mean ± SD. p < 0.05 was considered as significant. ( F ) H1975 and H661 cells were treated with 50 nM STS alone or together with AAT (2 mg/mL) for 18 h. Annexin V surface expression was determined by flow cytometry. All experiments were performed in duplicates and repeated three times. Each point represents a mean ± SD. p < 0.05 was considered as significant. NTP: non-target-pool siRNA and pCVM6 as control vector.

Article Snippet: For overexpression of SERPINA1 , cells were transfected with the pCMV6-SERPINA1 (SC119951, OriGene, Rockville, MD, United States) vector or with the pCMV6 (PS100020, OriGene) vector as a control using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Migration, Knockdown, Over Expression, Knock-Out, Expressing, Flow Cytometry, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: Alpha-1-Antitrypsin: A Novel Human High Temperature Requirement Protease A1 (HTRA1) Substrate in Human Placental Tissue

doi: 10.1371/journal.pone.0109483

Figure Lengend Snippet: HTRA1 candidate substrates identified in the placenta protein.

Article Snippet: A1AT was purified from pooled human plasma (ProteaImmun GmbH, Berlin, Germany), and purity was confirmed by SDS-PAGE using coomassie blue staining (≥95% of total protein). β-Casein was purified from bovine Milk (Sigma), and purity was confirmed by SDS-PAGE using coomassie blue staining (≥98% of total protein).

Techniques: Membrane

Journal: PLoS ONE

Article Title: Alpha-1-Antitrypsin: A Novel Human High Temperature Requirement Protease A1 (HTRA1) Substrate in Human Placental Tissue

doi: 10.1371/journal.pone.0109483

Figure Lengend Snippet: Details of the 2-D pattern of the HTRA1 substrate A1AT in the placenta protein sample after incubation in the absence (−HTRA1) and in the presence (+HTRA1) of HTRA1 (A). The images are small sections of the . The arrows indicate placenta A1AT (spots A2) and placenta A1AT fragments (Spots B1). On the right hand site: Sequences of A1AT identified by MS analysis (bold sequence) after tryptic in-gel digestion of the spots A2 and B1. The italic sequence indicates the signal peptide; the underlined sequence indicates the reaction center loop. (B) A1AT forms a stable complex with HTRA1 at high molecular weight range (spot B7); this was supported by MS measurements. Three replicates are shown in .

Article Snippet: A1AT was purified from pooled human plasma (ProteaImmun GmbH, Berlin, Germany), and purity was confirmed by SDS-PAGE using coomassie blue staining (≥95% of total protein). β-Casein was purified from bovine Milk (Sigma), and purity was confirmed by SDS-PAGE using coomassie blue staining (≥98% of total protein).

Techniques: Incubation, Sequencing, High Molecular Weight

Journal: PLoS ONE

Article Title: Alpha-1-Antitrypsin: A Novel Human High Temperature Requirement Protease A1 (HTRA1) Substrate in Human Placental Tissue

doi: 10.1371/journal.pone.0109483

Figure Lengend Snippet: Purified A1AT from human plasma was incubated with HTRA1 at 37°C for 17 hours. (A) 2-D gel of A1AT in the absence of HTRA1 incubation time 0 h and SDS-PAGE of A1AT and HTRA1. The A1AT preparation contains only very small amount of cleaved A1AT. (B) Incubation over a period of 17 hours in the absence (left site) and presence (right site) of HTRA1. After incubation with HTRA1 a significant amount of A1AT was cleaved. The incubation of plasma A1AT with HTRA1 showed similar fragmentation patterns as the incubation of placental A1AT with HTRA1 .

Article Snippet: A1AT was purified from pooled human plasma (ProteaImmun GmbH, Berlin, Germany), and purity was confirmed by SDS-PAGE using coomassie blue staining (≥95% of total protein). β-Casein was purified from bovine Milk (Sigma), and purity was confirmed by SDS-PAGE using coomassie blue staining (≥98% of total protein).

Techniques: Purification, Clinical Proteomics, Incubation, SDS Page

Journal: PLoS ONE

Article Title: Alpha-1-Antitrypsin: A Novel Human High Temperature Requirement Protease A1 (HTRA1) Substrate in Human Placental Tissue

doi: 10.1371/journal.pone.0109483

Figure Lengend Snippet: MALDI-Orbitrap-MS analysis as described in experimental procedures. A: Peptide A1AT(361–384) and generated fragment. Cleavage occurs after met381. B: Peptide A1AT(379–400) and generated fragment. Cleavage occurs again after met381. Further signals are sodium adduct (+22) and potassium adduct (+38). The image is representative of three independents experiments. The same incubation experiment was performed without addition of HTRA1 as negative control .

Article Snippet: A1AT was purified from pooled human plasma (ProteaImmun GmbH, Berlin, Germany), and purity was confirmed by SDS-PAGE using coomassie blue staining (≥95% of total protein). β-Casein was purified from bovine Milk (Sigma), and purity was confirmed by SDS-PAGE using coomassie blue staining (≥98% of total protein).

Techniques: Generated, Incubation, Negative Control