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Cancers 2020, 12, 2371 10 of 14 4.5. Immunoblotting Cellular proteins were extracted, separated by electrophoresis, transferred to nitrocellulose membranes, and probed, as described previously [16]. The antibodies used for western blotting were anti-LC3B (Novus, NB600-1384, Saint Charles, MI, USA; Cell Signaling Technology, 12741S, Danvers, MA, USA), anti-human SQSTM1 (Abcam, ab56416; Cambridge, MA, USA), and anti-AKTser473 (Cell Signaling Technology, 9271; Danvers, MA, USA). Membranes were stained with Ponceau S Red (Sigma, P-3504; ST-Louis, MO, USA) for protein visualization. After initial probing, membranes were stripped with Restore Plus western blot stripping buffer (Thermo Fisher Scientific, 46430; Waltham, MA, USA) and then re-probed with anti-alpha-tubulin as a loading control (Cell Signaling Technology, 3873; Danvers, MA, USA). Alternatively, cellular proteins were separated by electrophoresis in stain-free gels (Bio-Rad, 456-8124; Hercules, CA, USA) for direct visualization and quantification of the proteins. Densitometric analyses were conducted with the ImageLab software from BioRad (version 1.0; Hercules, CA, USA). Data are expressed in arbitrary units. 4.6. Tandem Sensor RFP-GFP-LC3B Expression We used the PremoTM autophagy tandem sensor RFP-GFP-LC3B BacMam 2.0 expression vector kit from Thermo Fisher (P36239; Waltham, MA, USA) and followed instructions that were provided by the manufacturer. In brief, the HNSCC cell lines were plated in 24-well plates containing coverslips at a density of 30,000 cells/well for SCC-9 and 40,000 cells/well for FaDu. The day after, the cells were infected with baculovirus at a multiplicity of infection of 30 in complete medium. After overnight incubation, cells were exposed to experimental conditions for 24 h unless otherwise stated. Cells were then fixed with formalin for 15 min., washed with PBS, and coverslips were mounted onto slides with Prolong Gold® anti-fade reagent with DAPI (Thermo Fisher, P36935; Waltham, MA, USA). Cells were observed using a confocal microscope (Leica TCS SP5 MP; Concord, ON, Canada). Three pictures (64 times magnification), each capturing approximately 15–30 cells, were taken per condition. The Image J software with a macro adapted from Daniel J. Shiwarski, Ruben K. Dagda and Charleen T. Chu (https://imagejdocu.tudor.lu/plugin/analysis/colocalization_analysis_macro_for_red_and_green_ puncta/start) was used for quantification. 4.7. Reagents Buparlisib (NVP-BKM120) (11587) was purchased from Cayman Chemical, Ann Arbor, MI, USA; Omipalisib (GSK2126458) (S2658) from Selleckchem, Houston, TX, USA; Rapamycin (R0161) from LKT Labs, St. Paul, MN, USA; Bafilomycine A1 (B1793), Chloroquine (C6628), and phosphatase inhibitor cocktail 2 (5726) from Sigma, ST-Louis, MO, USA; protease inhibitor cocktail 3 (535140) from Calbiochem, San Diego, CA, USA; and acridine orange (A-1301) from Thermo Fisher, Waltham, MA, USA. All other reagents were from Sigma Chemicals, ST-Louis, MO, USA. 4.8. Statistical Analysis Comparisons of PD among different treatment groups were made using Kruskal–Wallis one-way analysis of variance (ANOVA) for matched data with post-hoc Tukey HSD test with the GraphPad Prism software version 8 (https://www.graphpad.com/scientific-software/prism). Bar graphs were presented as mean ± standard deviation (SD). Values of * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 were considered to be significant. * p without underscore bar represents a significant difference with the control (DMSO vehicle). Other results, expressed as mean ± SD, were analyzed by unpaired Student’s t-test. Values of * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 were considered significant. * p without underscore bar represents a significant difference with the control (DMSO vehicle).PDF Image | Dual Inhibition of Autophagy Pathway as a Therapeutic Strategy
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