Journal: Microbial cell factories

Article Title: Secretion of tumoricidal human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by recombinant Lactococcus lactis: optimization of in vitro synthesis conditions.

doi: 10.1186/s12934-018-1028-2

Figure Lengend Snippet: Fig. 9 Selectivity of hsTRAIL-induced death to cancer cells. HCT116 cells and human cardiac fibroblasts were incubated for 48 h with increasing concentration of hsTRAIL present in supernatant from broth culture of L. lactis (hsTRAIL+). As controls in experimental setup were used: corresponding volumes of supernatants from broth culture of L. lactis (hsTRAIL−)—negative control; corresponding concentrations of recombinant human TRAIL (Peprotech)—positive control. Viability of cancer cells and non-malignant, cardiac fibroblasts was assessed by MTS assay. Results are presented as % of viability of cells incubated in standard culture medium only. Secreted hsTRAIL remained non-cytotoxic to fibroblasts (Fig. 9a), while decreased viability of cancer cells in a dose-dependent manner (Fig. 9b). Table below—concentration of hsTRAIL [ng/ml] in specified volume of supernatant. The bars indicate the mean value ± SD of three independent experiments, each performed in triplicates. Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparison post-test. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Human primary proliferating cardiac fibroblasts were obtained from Cell Applications, Inc. (San Diego, CA).

Techniques: Incubation, Concentration Assay, Negative Control, Recombinant, Positive Control, MTS Assay, Comparison

Journal: Cardiovascular Research

Article Title: Human epicardial cell-conditioned medium contains HGF/IgG complexes that phosphorylate RYK and protect against vascular injury

doi: 10.1093/cvr/cvv168

Figure Lengend Snippet: HGF is a key vaso-protective factor in EPI CdM. (A) EPI CdM protected cultured primary human coronary artery endothelial cells for 48 h under simulated ischemia, as measured by MTS assay (n = 5 donors). Data are mean ± S.D. (B) EPI CdM protected cultured human microvascular endothelial cells for 48 h under simulated ischemia, as measured by CyQuant assay (n = 3 donors). Data are mean ± S.D. In both experiments, survival of cells in growth medium (GM) was considered as 100%. (C) Receptor tyrosine kinase (RTK) array demonstrates 30 min exposure to 1× EPI CdM induces phosphorylation of multiple growth factor receptors of coronary artery endothelial cells compared with MEM treatment (n = 2). (D) HGF ELISA data are for 5 different EPI CdM donors (D1–D5). Measurements are in duplicate. (E) Pull down (PD) of HGF from EPI CdM with an HGF-specific antibody (2 µg/ml) decreased EPI CdM-mediated protection of microvascular endothelial cells during simulated ischemia (48 h) compared to PD with a non-specific IgG antibody (*P ≤ 0.05 Control IgG vs. Anti-HGF). In contrast, PD of SDF-1, ANG1 or VEGFA from EPI CdM did not alter its protection of microvascular endothelial cells (all antibodies used at 2 µg/ml) (n = 3). Cell numbers were determined by CyQuant assay (dye binding of nucleic acids). Data are mean ± S.D. One way ANOVA for A,B, and E: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Scale bar = 100 µM.

Article Snippet: EPI CdM protects human cardiac endothelial cells during simulated ischaemia To investigate EPI CdM-mediated protection of cardiac endothelial cells, human cardiac endothelial cells were purchased from Lonza (Catalog # CC-2585 and CC-7030, Passage 1).

Techniques: Cell Culture, MTS Assay, CyQUANT Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Control, Binding Assay

Journal: Cardiovascular Research

Article Title: Human epicardial cell-conditioned medium contains HGF/IgG complexes that phosphorylate RYK and protect against vascular injury

doi: 10.1093/cvr/cvv168

Figure Lengend Snippet: HGF/IgG complexes enhance vascular protection by interacting with RYK. (A) Stable producer cell lines and purification of bioactive recombinant human HGF. (Left, top) Phase contrast images of HEK 293 cells with genomic integration of pIRES-puro vector expressing human HGF-His tag-F2A-GFP. (Left, bottom) Epifluorescence image showing GFP expression (FITC channel). (Right) Gel electrophoresis illustrating purification of HGF. Lane 1, electrophoresis of eluted material under non-reducing conditions demonstrates purity and molecular weight of single chain pro-HGF (90 kDa). Gel stained with Coomassie Brilliant Blue. Lane 2, purified protein run under reducing conditions (beta-mercaptoethanol) demonstrates the active heterodimer of human HGF (see bands at 65 kDa and 32 kDa). (B) CyQuant assay demonstrates enhanced protective effects of HGF/IgG complexes when compared with free HGF (alone) or free HGF with IgG (Both free, unconcentrated factors). For greater detail, please refer to Methods. Cell number was assayed after 48 h of simulated ischemia (MEM, 69 ± 24.7% of HGF; free HGF alone, 100 ± 7.3%; free HGF with IgG [Free], 114 ± 3.1% of HGF; HGF with IgG [Complex], 169 ± 4.0% of HGF [n = 4 for all]). Free HGF with IgG (Free) conferred protection (P ≤ 0.01, vs. MEM). However, HGF/IgG complexes (Complex) protected better than did free HGF with IgG (Free) (P ≤ 0.001). (C) Treatment of coronary endothelial cells with HGF/IgG complexes (Complex, top), free HGF and IgG (Free, middle) or MEM (bottom) affects the level of phosphorylation for RYK, but not c-Met. Positive control signal (pos-ctrl) has been included for each membrane to compare for normalization (n = 2). (D) Blocking RYK reduced protection conferred by HGF/IgG complexes (P ≤ 0.01, n = 4) but not protection by free HGF with IgG (P = 0.09, n = 4). Note: coronary artery endothelial cells were incubated under simulated ischemia for 24 h. (E) Intra-arterial treatment with HGF/IgG complexes significantly decreased FITC-albumin extravasation in the LV wall of rats at 24 hr after reperfusion (MEM, n = 4; free HGF with IgG [Free], n = 8; HGF/IgG complexes [Complex], n = 11; P ≤ 0.01). (F and F′) Epifluorescent image showing localization of His-HGF outside of blood vessels (by anti-His antibody) in animals treated with free HGF (F) and complex HGF (F′). HGF/IgG complexes were localized to large blood vessels (F and F′) and the capillary network at the borders of infarcted regions (F″). We confirmed a similar staining pattern in hearts of 3 different animals. Note: the presence of HGF not localized to capillaries is likely indicative of leakage from damaged or dead blood vessels in the capillary bed. Data are mean ± S.D. One way ANOVA for B,D, and E: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Scale bar = 100 µM.

Article Snippet: EPI CdM protects human cardiac endothelial cells during simulated ischaemia To investigate EPI CdM-mediated protection of cardiac endothelial cells, human cardiac endothelial cells were purchased from Lonza (Catalog # CC-2585 and CC-7030, Passage 1).

Techniques: Purification, Recombinant, Plasmid Preparation, Expressing, Nucleic Acid Electrophoresis, Electrophoresis, Molecular Weight, Staining, CyQUANT Assay, Phospho-proteomics, Positive Control, Membrane, Blocking Assay, Incubation