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: Lactococcus lactis -derived hsTRAIL-induced death of human colon cancer HCT116 cells in vitro. HCT116 cells were incubated for 48 h with increasing concentrations of hsTRAIL in supernatants from broth culture of L. lactis (hsTRAIL+). The following controls in experimental setup were used: corresponding volumes of supernatants of L. lactis (hsTRAIL−)—negative control; corresponding concentrations of recombinant human TRAIL (Peprotech)—positive control. Viability of cancer cells was assessed by MTS assay. Results are presented as % of viability of cells incubated in standard culture medium only. hsTRAIL present in supernatant from L. lactis (hsTRAIL+) broth culture decreased viability of cancer cells in a dose-dependent manner, comparable to corresponding concentrations of positive control. Table below—concentration of TRAIL [ng/ml] in specified volume of supernatant or concentration of standard TRAIL formulation (rhTRAIL, Peprotech). 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: As positive control, standard TRAIL formulation (rhTRAIL, Peprotech) in corresponding concentration was used ( e ).

Techniques: Derivative Assay, In Vitro, Incubation, Negative Control, Recombinant, Positive Control, MTS Assay, Concentration Assay, Formulation, Comparison

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: Induction of apoptosis of colon cancer HCT116 cells by hsTRAIL from supernatant of L. lactis (hsTRAIL+). HCT116 cells were treated with supernatants from broth cultures of L. lactis (hsTRAIL±) bacteria and rhTRAIL (positive control) in a corresponding dose of 100 ng/ml for 48 h and then stained with FITC-Annexin V and PI to determine cancer cells apoptosis. HCT116 turned apoptotic after treatment with supernatant of L. lactis (hsTRAIL+) bacteria ( a , d ) in contrast to supernatant of L. lactis (hsTRAIL−) (negative control; c ) or medium ( b ). As positive control, standard TRAIL formulation (rhTRAIL, Peprotech) in corresponding concentration was used ( e ). Dot-plots. FL1-H—FITC; FL2-H—PI. The bars ( a ) indicate the mean value ± SD of three independent experiments, each performed in triplicates. Differences between the groups were calculated using two-way ANOVA with Tukey’s multiple comparisons post-test. *p < 0.05

Article Snippet: As positive control, standard TRAIL formulation (rhTRAIL, Peprotech) in corresponding concentration was used ( e ).

Techniques: Bacteria, Positive Control, Staining, Negative Control, Formulation, Concentration Assay

Journal: Oncotarget

Article Title: The antidepressant fluoxetine induces necrosis by energy depletion and mitochondrial calcium overload

doi: 10.18632/oncotarget.13689

Figure Lengend Snippet: A . Confocal microscopy images of HeLa cells, transfected with a construct enabling transient expression of a fusion protein [Pleckstrin Homology (PH) domain of PLC] / eGFP fluorescent protein, allowing to locate membrane PIP 2 , as well as cytosolic IP 3 . Fluorescence was measured in zones of interest delimited by rectangles, corresponding to membrane (rectangle “1”) and cytosolic regions (rectangle “2”), respectively. B and C . Time course of fluorescence emission, at the membrane level (black trace) and cytoplasm (red or blue trace): B - To induce IP 3 translocation from membrane to cytoplasm, rhTRAIL (100ng/mL) was injected when indicated by arrow. C - Fluoxetine (100μM) does not induce IP 3 translocation. D . Jurkat cells were loaded with FuraPE3-AM Ca 2+ probe. Cells were either PLCγ deficient (PLC def) or reconstituted with a functional PLCγ (PLC rec). E . Jurkat cells were loaded with FuraPE3-AM, and fluorescence ratios were recorded in the presence of 2mM of external Ca 2+ (black trace). Ratios were also recorded in the presence of the IP 3 Rs inhibitors xestospongin C (500nM) and 2-APB (44μM, green and red traces, respectively). F . Quantification of results obtained in E: histogram bars represent the area under the curve (AUC), expressed in arbitrary units (A.U.), for control (CTL), xestospongin C and 2-APB experiments. G . Jurkat cells were loaded with FuraPE3-AM, and fluorescence ratios were recorded in the presence of 10μM dantrolene (inhibitor of RyR1 and RyR3 isoforms, green trace), 10μM U73122 (inhibitor of PLCβ, purple trace) and 5μM Ned19 (TPC inhibitor, blue trace). H . Quantification of results obtained in G: histogram bars represent the area under the curve (AUC), expressed in arbitrary units (A.U.), for control (CTL), dantrolene, U73122 and Ned19 experiments.

Article Snippet: Oligomycin and rhTRAIL were obtained from Merck Millipore (Fontenay sous Bois, France) and AdipoGen (Liestal, Switzerland), respectively.

Techniques: Confocal Microscopy, Transfection, Construct, Expressing, Membrane, Fluorescence, Translocation Assay, Injection, Functional Assay, Control

Journal: Biotechnology progress

Article Title: A Simplified Method for the Efficient Purification and Refolding of Recombinant Human TRAIL

doi: 10.1002/btpr.3007

Figure Lengend Snippet: Expression, purification and refolding of recombinant human TRAIL (rhTRAIL).

Article Snippet: Cells were incubated with purified rhTRAIL and commercial rhTRAIL (Enzo Life Science) at 5, 10, 20 and 40 ng/mL for 2 hours before being collected and stained with Annexin V and Propidium Iodide (PI).

Techniques: Expressing, Purification, Recombinant

Journal: Biotechnology progress

Article Title: A Simplified Method for the Efficient Purification and Refolding of Recombinant Human TRAIL

doi: 10.1002/btpr.3007

Figure Lengend Snippet: Samples were analyzed in 12% Tris-Tricine SDS-PAGE and stained with Coomassie Bright Blue. Lane 1: molecular weight marker. Lane 2: total cell lysate. Lanes 3: fraction washed off the Ni-NTA column by the lysis buffer containing 30 mM imidazole. Lane 4: fraction of purified rhTRAIL eluted by the elution buffer containing 300 mM imidazole. The arrow indicates the rhTRAIL protein.

Article Snippet: Cells were incubated with purified rhTRAIL and commercial rhTRAIL (Enzo Life Science) at 5, 10, 20 and 40 ng/mL for 2 hours before being collected and stained with Annexin V and Propidium Iodide (PI).

Techniques: SDS Page, Staining, Molecular Weight, Marker, Lysis, Purification

Journal: Biotechnology progress

Article Title: A Simplified Method for the Efficient Purification and Refolding of Recombinant Human TRAIL

doi: 10.1002/btpr.3007

Figure Lengend Snippet: All the samples were diluted 10 times and analyzed in 12% Tris-Tricine SDS-PAGE and stained with Coomassie Bright Blue. Lane 1: molecular weight marker. Lane 2: denatured rhTRAIL purified by IMAC. Lanes 3: rhTRAIL renatured by stepwise dilution. Lane 4: rhTRAIL after dialysis to remove urea completely. Lane 5: rhTRAIL after concentration with protein concentrator. The arrow indicates the TRAIL protein.

Article Snippet: Cells were incubated with purified rhTRAIL and commercial rhTRAIL (Enzo Life Science) at 5, 10, 20 and 40 ng/mL for 2 hours before being collected and stained with Annexin V and Propidium Iodide (PI).

Techniques: SDS Page, Staining, Molecular Weight, Marker, Purification, Concentration Assay