PDF Publication Title:
Text from PDF Page: 009
Cancers 2020, 12, 2371 9 of 14 allowing for better therapeutic choices for improved treatment outcomes. This will be possible with tumor genetic sequencing for oncogene mutations, but also with ex-vivo testing of inhibitors on tumor fragments before chemotherapy treatment [52]. Investigation into the long-term inhibitory effects of these pathways in normal or cancer cells are still at an early stage and require further study for their safe use in personalized therapies. 4. Materials and Methods 4.1. Cell Culture Human squamous carcinoma cell lines of the oral tongue (SCC-4, SCC-9, SCC-25), hypopharynx (FaDu), and of a metastasis from the pharynx (Detroit 562) were purchased from the American Type Culture Collection (Manassas, VA, USA) (CRL-1624, CRL-1629, CRL-1628, HTB-43, and CCL-138). The UM-SCC-22A (hypopharynx) cell line was purchased from EMD Millipore Corporation (Burlington, MA, USA) (SCC076). The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (GibcoTM, 11995-065; Waltham, MA, USA) supplemented with 10% fetal bovine serum (Wisent, 088150; Saint-Jean-Baptiste, QU, Canada), 1% penicillin/streptomycin (Wisent, 450-201-EL; Saint-Jean-Baptiste, QU, Canada), and 1 times non-essential amino acids (GibcoTM, 11140-050; Waltham, MA, USA), and they were used before the tenth passage from the first thaw after reception. All of the cell lines are HPV-negative. 4.2. IncuCyte® Live-Cell Analysis for Cell Proliferation and Bliss Synergy Score Cells were seeded in 96-well plates, 100 μL per well, and allowed to adhere overnight. SCC-4, SCC-9, and SCC-25 were seeded at a density of 3500 cells/well; FaDu and UM-SCC-22A at 5000 cells/well; and, Detroit 562 at 10,000 cells/well. The following day, 100 μL of media containing 2 times concentration of vehicle or inhibitor or combination of inhibitors was added in triplicates. PI3Ki stock solutions were prepared in 100% dimethyl sulfoxide (DMSO). Working solutions were prepared fresh before addition to the cell media, such that final DMSO concentrations were kept constant in both control and treated cells. Proliferation assays consisted of longitudinal imaging of the cell layer and confluence evaluation with the IncuCyte S3 system (Essen Bioscience, Ann Arbor, MI, USA). Phase contrast images of the full well were acquired at 4-h intervals for 72 h at 4 times magnification. The proliferation curves were generated using the confluence evaluation algorithm of the IncuCyte S3 software. PD was calculated using this formula: log2 (confluence at 72 h/confluence at 0 h). 4.3. Bliss Synergy Score Calculation The cells were incubated, as described above, with vehicle DMSO or different concentrations of the inhibitors, alone or in combination. Real-time proliferation was recorded for 3–4 days at 4-h intervals with IncuCyte S3 (Essen Bioscience, Ann Arbor, MI, USA). Area under the curve (AUC) with baseline correction was calculated using GraphPad version 8 (https://www.graphpad.com) when control conditions reached 75–80% confluence, as evaluated with the IncuCyte S3 software. AUC results were expressed as % proliferation versus control and formatted in a matrix for analysis with SynergyFinder software [46]. A Bliss synergy score over 1 indicates synergy. 4.4. Acridine Orange Staining Cells were seeded in 96-well plates, 100 μL per well, and allowed to adhere overnight. The following day, 100 μL of media containing 2 times the concentration of vehicle or inhibitor or inhibitor combinations was added in triplicates. The cells were incubated for 24 h and acridine orange was added at a final concentration of 5 μg/mL for 15 min. Images of phase contrast, and green and red fluorescence were acquired with the IncuCyte S3 system (Essen Bioscience, Ann Arbor, MI, USA) at 20 times magnification of two images per well.PDF Image | Dual Inhibition of Autophagy Pathway as a Therapeutic Strategy
PDF Search Title:
Dual Inhibition of Autophagy Pathway as a Therapeutic StrategyOriginal File Name Searched:
cancers-12-02371.pdfDIY PDF Search: Google It | Yahoo | Bing
Cruise Ship Reviews | Luxury Resort | Jet | Yacht | and Travel Tech More Info
Cruising Review Topics and Articles More Info
Software based on Filemaker for the travel industry More Info
The Burgenstock Resort: Reviews on CruisingReview website... More Info
Resort Reviews: World Class resorts... More Info
The Riffelalp Resort: Reviews on CruisingReview website... More Info
| CONTACT TEL: 608-238-6001 Email: greg@cruisingreview.com | RSS | AMP |