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Cancers 2018, 10, 144 12 of 15 previously [38]. The supernatant from each plate was collected for measurement of absorbance at a wavelength of 570 nm. The results are expressed as the percentage of live cells. For control infection, we used cell-line-specific media alone without virus or DMSO instead of TMZ. For combined therapies, cells were treated with OAdmCherry or AdGFP at a MOI concentration of 2.5 or TMZ of 0.4 mM, respectively. 4.4. Adenovirus Titer Assay Cells were infected with OAdmCherry alone or were treated as described in the previous section; 72 h after treatment, supernatants were collected and centrifuged for 10 min at 14,000 rpm. The supernatants were then transferred to a new tube to eliminate cell debris and/or cells in suspension that may have contained Ad’s. Supernatants were diluted serially by using the median tissue culture infective dose, the amount of a pathogenic agent that would produce pathological change in 50% of cell cultures inoculated (TCID50), or by using the end-point dilution method with HEK-293 cells seeded on 96-well plates. Briefly, HEK 293 cells were seeded in 96-well plates at a density of 103 (cells per well) and were then infected with 10-fold serially diluted viruses. CPE was recorded and scored after incubation for 7 days. The reduction percentage in virus titer was calculated by the following formula: reduction % = [(titer of control group − titer of experimental group)/titer of control group] × 100% [16,41]. 4.5. Western Blot Analysis Cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer, as described previously [42]. Cell lysates were centrifuged, and the protein concentration was determined by a Pierce bicinchoninic acid assay (BCA) protein kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of cellular protein were electrophoresed on 10–12% SDS–polyacrylamide gels and transferred to Hybond-Polyvinylidene fluoride (PVDF) () membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The primary antibodies used were rabbit anti-LC3 polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA), mouse anti-adenovirus type 5 E1A (BD Pharmingen, San Diego, CA, USA), and rabbit anti-human actin (Sigma-Aldrich, St. Louis, MO, USA). Next, the membranes were incubated with anti-mouse immunoglobulin (Ig) or anti-rabbit Ig, peroxidase-linked, species-specific whole antibody (Thermo Fisher Scientific, Waltham, MA, USA). (Electrochemiluminescence) (ECL) reagents were used to detect the signals according to the manufacturer’s instructions (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The scanned band intensities were quantified using Gel-pro Analyzer 4.0 software (Media Cybernetics, Rockville, MD, USA) according to the manufacturer’s instructions. Densitometric values for each band were expressed as integrated optical density (I.O.D.) and were normalized to actin expression. 4.6. GFP-LC3 Puncta Plasmid vector containing green fluorescent protein linked to microtubule-associated protein 1 LC3 was used to detect autophagosome formation in TNBC cell lines [28]. At 24 h post transfection, cells were treated with TMZ at 0.4 mM or OAdmCherry at a MOI concentration of 2.5 alone or in combination. At 48 h post treatment, cells were examined under a fluorescence microscope. Cells were classified as having a predominantly diffuse GFP stain or having numerous punctate structures representing autophagosomes. Images were taken at 40x magnification with the EVOS FL Imaging System (Thermo Fisher Scientific, Waltham, MA, USA) under 357/44 and 447/60 nanometers (nm) excitation and emission visualization, respectively. The percentage of cells with GFP puncta were calculated as the proportion of cells with GFP puncta divided by the total number of GFP expressing cells.PDF Image | Temozolomide Enhances Triple-Negative Breast Cancer Virotherapy
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