Viktor3

To determine cell viability, CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) was used according to manufacturer’s instructions. Briefly, 500 cells were seeded per well in a 96-well plate, in triplicate. Cells were incubated for 24 h at 37°C, 5% CO₂. Drugs were applied at a range of concentrations (0 μM – 5 mM) and incubated for 48 h. The plate was equilibrated at room temperature for 30 min and 100 μl of CellTiter-Glo® reagent was added to each well. Luminescence was recorded by a luminometor (Viktor3, PerkinElmer, Massachusetts, USA). IC₅₀ values were calculated from three replicates using GraphPad Prism version 5 software (GraphPad Software Inc., San Diego, CA).

IC50 studies in the glioma cell lines upon drug exposure were performed using the sulforhodamineB (SRB) assay. Briefly, 48 h after exposure to nine increasing concentrations of deoxynyboquinone (2 nM–15 µM), culture medium was removed and 100 µL of 10% trichloroacetic acid was added to the wells to fix cells for 1 h at 4 °C. Cells were then washed twice with distilled water and stained with 100 µL of 0.4% SRB in 1% acetic acid during 30 min, light-protected, and washed twice with 1% acetic acid. SRB was then solubilized in Tris base (10 mM; pH 10.0) and 540 nm-optical density was determined using a microplate reader (Perkin Elmer Viktor 3).

Plasma level of stromal cell-derived factor-1 (porcine SDF-1α ELISA Kit; Neoscientific, Germany), chemokine (C-X-C motif) receptor 4 (pig CXCR4 ELISA Kit; Abbexa, UK), 72kDa isoform of matrix metalloproteinase-2 protein (porcine MMP-2 ELISA Kit; MyBioSource, CA, USA), fibroblast growth factor-2 (porcine FGF-2 ELISA Kit; Neoscientific, Germany), and vascular endothelial growth factor (porcine VEGF ELISA Kit; Neoscientific, Germany) were detected using commercial ELISA kits, according to the manufacturer’s instructions. Tumor necrosis factor alpha (Porcine TNFα Quantikine ELISA Kit; R&D Systems, MN, USA), and interleukin-8 (pig IL-8 ELISA Kit; Abcam, UK) were detected from serum, according to the manufacturer’s instructions.
Absorbance readings at wavelength 450nm were performed on the automated plate reader VIKTOR3 (Perkin Elmer, MA, USA), and the resulting values were determined by interpolation from a standard curve. Measurements were performed in duplicates. Plasma or serum levels of markers were measured at baseline, 3d post MI and at 1 month FUP.

Activation of caspase-2, caspase-8, caspase-9 and caspase-3 on indicated drug treatments was determined using a caspase profiling plate (Clontech lab., CA, USA) according to the manufacturer’s instructions. Briefly, cells were grown until 60-70% confluent, then treated with either carprofen or mavacoxib (50 μM and 100 μM), and incubated for 48 h at 37°C, 5% CO₂. Untreated cells were also prepared as a control for each cell line. After treatment, 2 × 10⁵ cells were prepared per well, lysed in cold 1× Cell Lysis Buffer (50 μl/2 × 10⁵ cells) and incubated for 10 min on ice. The plates were preincubated with 50 μl of 2× Reaction buffer (mixed with 0.1 M DTT) to each well for 5 min at 37°C, 5% CO₂. Cell lysates were transferred to each well and incubated for 2 h at 37°C, 5% CO₂. Caspase activities were read using a fluorescent plate reader (excitation: 380 nm, emission: 460 nm) (Viktor3, PerkinElmer, Massachusetts, USA).

Cell numbers on the scaffolds were determined using the CyQuant DNA assay (Molecular Probes, Oregon, USA) following Proteinase K digestion. At the designated time points, cell culture media was aspirated off and the cell-scaffold constructs were washed thrice with PBS and frozen at −80 °C until further processing. Samples were then digested at 56 °C in a Tris-EDTA buffered solution containing 1 mg/ml Proteinase K, 18.5 μg/ml pepstatin A and 1 μg/ml iodoacetamide (Sigma). DNA quantification was performed following manufacturer’s instructions using a sepctrofluorometer (Viktor3, Perkin Elmer).

Cells were trypsinised into single cells and seeded in quadruplet in opaque 96-well plates (Corning, CA, USA) at 500 cells/well. Serial dilutions of doxorubicin, gefitinib, or ionising radiation were added to the appropriate cells the following day or as indicated. Dose-response curves were generated 48 hours after exposure. Cytotoxicity was measured using the CellTiterGlo® Luminescent Cell Viability Assay (Promega, Madison, USA), which quantifies the number of viable cells in culture based on quantification of ATP present. Luminescence was recorded by luminometer (Viktor3, PerkinElmer, Massachusetts, USA). Data was averaged and normalized against the average signal of untreated/vehicle control treated samples.

Cells were plated on 96-well tissue-culture microtiter plates at a density of 4 × 10⁴ cells per well. The miR-21 inhibitor, and the respective controls were transfected to the cells for 6 hours, then the culture medium was removed, and cells were maintained with RPMI 1640 supplemented with 8% FBS. Next day, the cells were treated with the two drugs for 24 hours at various concentrations (cabozantinib at 1.4 µM for MZ-CRC-1 and 9.7 µM for TT; vandetanib at 10 µM for both cell lines). We measured the inhibitory effect on cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich). Then the culture media were removed and 100 µL DMSO was added to dissolve the formazan crystals, and absorbance was measured at 595 nm (Viktor 3 Perkin-Elmer). The signal intensity was then presented as a percentage relative to the control. Experiments were run in triplicate and repeated three times.