recombinant human rh trail  (R&D Systems)

Journal: Oncotarget

Article Title: Suppression of SOCS3 enhances TRAIL-induced cell growth inhibition through the upregulation of DR4 expression in renal cell carcinoma cells

doi: 10.18632/oncotarget.25851

Figure Lengend Snippet: ACHN and 786-O cells were treated with recombinant human TRAIL (0-1000 ng/mL) and anti-6X histidine mAb (10 μg/mL). The relative absorbances (mean ± SE) compared with non-treated cells, as a measure of cell viability, obtained from the WST-1 assay are shown. Significant differences were observed at doses of 12.3 ng/mL ( p < 0.05) and over ( p < 0.01).

Article Snippet: Recombinant human (rh) TRAIL and anti-6X histidine mAb were purchased from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Recombinant, WST-1 Assay

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Macrophage Elastase Induces TRAIL-mediated Tumor Cell Death through Its Carboxy-Terminal Domain

doi: 10.1164/rccm.201606-1150OC

Figure Lengend Snippet: C-terminal domain (CTD) and SR20 induce non–small cell lung cancer cell line apoptosis but do not affect cell cycle progression. A549, 91T, H1650, and 201T cells were untreated (vehicle) or treated with CTD, dimethyl sulfoxide (DMSO), or SR20 (20 μg/ml or 50 μg/ml). Cells were labeled with Annexin V after (A) CTD (n = 2–15 replicates per group) or (B) SR20 (n = 4 replicates per group) treatment, and the percentages of Annexin V+ cells were quantified by flow cytometry. (C) Representative flow cytometry plots of A549 cells labeled with Annexin V and 7-AAD after SR20 treatment (plots are representative of four independent experiments). (D) A549, (E) 91T, (F) H1650, and (G) 201T cells were stained with propidium iodide after CTD (n = 2–4 replicates per group) or SR20 (n = 3–4 replicates per group) treatment, and the percentages of cells in the G0-G1 or G2-M phases of the cell cycle were quantified by flow cytometry. (H) Representative histograms of A549 cells labeled with propidium iodide after DMSO or SR20 treatment (plots are representative of four independent experiments). Values represent the percentages of cells in the respective phases of the cell cycle (G0-G1 or G2-M). The bars delineate the propidium iodide levels associated with the cell cycle phases. Data are mean ± SD. P values were calculated by two-sided independent sample Student’s t test. 7-AAD = 7-aminoactinomycin D.

Article Snippet: Cells were treated with 20–50 μg/ml SR20 (in 10% dimethyl sulfoxide; Sigma-Aldrich, St. Louis, MO), 50 μg/ml CTD, 50 μg/ml MMP9 CTD, 100 μg/ml CAT, or 30–100 ng/ml recombinant human (rh) tumor necrosis factor–related apoptosis-inducing ligand (TRAIL; R&D Systems, Minneapolis, MN) for 1 hour in serum-free Dulbecco’s modified Eagle medium and then incubated in serum-free media for 24–72 hours.

Techniques: Labeling, Flow Cytometry, Staining

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Macrophage Elastase Induces TRAIL-mediated Tumor Cell Death through Its Carboxy-Terminal Domain

doi: 10.1164/rccm.201606-1150OC

Figure Lengend Snippet: C-terminal domain (CTD) induces the expression of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and death receptors 4 (DR4). A549 cells were treated with 50 μg/ml CTD for 1 hour. (A) Relative mRNA expression of TRAIL and DR4 normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) control at 5 hours after CTD treatment (n = 4 means of independent experiments). (B) Representative Western blot of TRAIL, DR4, and GAPDH endogenous control protein 48 hours after CTD treatment. (C) Western blot representative of three independent experiments and (D) quantitative ELISA for soluble TRAIL (sTRAIL) in culture media at 48 hours after CTD treatment (n = 3 means of independent experiments). (E) Confocal microscopy of CTD-treated A549 cells stained with rhodamine-phalloidin (red), 4′,6-diamidino-2-phenylindole (DAPI; blue), and anti–matrix metalloproteinase-12 CTD (green). Images are representative of three independent experiments. Images were captured at ×100; scale bars are 10 μm. (F) A549 cytoplasmic and nuclear extracts were prepared at 0, 30, and 60 minutes after 50 μg/ml CTD treatment. Western blots are representative of three independent experiments. (G) A549 cells were cotransfected with pRL-CMV encoding Renilla luciferase and either pGL2-Basic, pGL2-Control, or pGL2-TRAIL encoding firefly luciferase and treated with 50 μg/ml CTD for 24 hours. Firefly luciferase activity was normalized to Renilla luciferase activity and expressed relative to vehicle-treated control animals (n = 6 replicates per group). Data are mean + SD. P values were calculated by two-sided independent sample t test. AU = arbitrary units; C = cytoplasmic; N = nuclear.

Article Snippet: Cells were treated with 20–50 μg/ml SR20 (in 10% dimethyl sulfoxide; Sigma-Aldrich, St. Louis, MO), 50 μg/ml CTD, 50 μg/ml MMP9 CTD, 100 μg/ml CAT, or 30–100 ng/ml recombinant human (rh) tumor necrosis factor–related apoptosis-inducing ligand (TRAIL; R&D Systems, Minneapolis, MN) for 1 hour in serum-free Dulbecco’s modified Eagle medium and then incubated in serum-free media for 24–72 hours.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Staining, Luciferase, Activity Assay

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Macrophage Elastase Induces TRAIL-mediated Tumor Cell Death through Its Carboxy-Terminal Domain

doi: 10.1164/rccm.201606-1150OC

Figure Lengend Snippet: C-terminal domain (CTD) induces tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-dependent apoptosis and TRAIL sensitization. A549 cells were treated with 50 μg/ml CTD for 1 hour, and experiments were conducted 48 hours later. (A) A549 cells were preincubated in the absence or presence of recombinant human DR4: IgG Fc chimera (rhDR4:Fc) for 30 minutes, continuing into CTD incubation, and [3H]-thymidine incorporation was measured (n = 3–4 replicates per treatment). (B) A549 cells were treated with CTD or 100 μg/ml catalytic domain. Representative Western blot of caspase (Casp) 3 and glyceraldehyde phosphate dehydrogenase (GAPDH). (C) A549 cells were preincubated with or without anti-TRAIL antibody, continuing into CTD incubation. Representative Western blot of Casp3 and GAPDH. (D) Representative images of vehicle and CTD-treated cells stained for TUNEL (scale bars are 100 μm). (E) TUNEL-positive cells were quantified as a percentage of total cells (n = 3 means of independent experiments). (F) Representative Western blot for Casp3 and GAPDH in CTD-treated cells plus or minus rhDR4:Fc. (G) Representative Western blot for Casp3, -8, -9 and GAPDH protein in cells treated with CTD in the presence or absence of Casp8 blocking peptides or Casp9 blocking peptides. (H) A549 cells were treated with CTD, 100 ng/ml rhTRAIL, or CTD plus 30 ng/ml rhTRAIL. Representative Western blot for phospho-Bad (pBad), Bad, Bcl-xL, Bcl-xS, XIAP, and GAPDH. (I) Representative Western blot for AKT, NFκB p50, p44/42 MAPK, and GAPDH. (J) Representative Western blot for c-FLIP and Casp3 in cells treated with CTD. Western blots are representative of three to six independent experiments. Data are mean + SD. P values were calculated by two-sided independent sample Student’s t test. Bad = Bcl2-associated death promoter; Bcl-xL = B-cell lymphoma–extra large; CAT = catalytic domain; c-FLIP = cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein; DPM = disintegrations per minute; DR4 = death receptor 4; MAPK = mitogen-activated protein kinase; NFκB = nuclear factor κ-light-chain-enhancer of activated B cells; PBS = phosphate-buffered saline; XIAP = X-linked inhibitor of apoptosis.

Article Snippet: Cells were treated with 20–50 μg/ml SR20 (in 10% dimethyl sulfoxide; Sigma-Aldrich, St. Louis, MO), 50 μg/ml CTD, 50 μg/ml MMP9 CTD, 100 μg/ml CAT, or 30–100 ng/ml recombinant human (rh) tumor necrosis factor–related apoptosis-inducing ligand (TRAIL; R&D Systems, Minneapolis, MN) for 1 hour in serum-free Dulbecco’s modified Eagle medium and then incubated in serum-free media for 24–72 hours.

Techniques: Recombinant, Incubation, Western Blot, Staining, TUNEL Assay, Blocking Assay, Saline