Computational protocol: 2-Methoxyestradiol enhances radiosensitivity in radioresistant melanoma MDA-MB-435R cells by regulating glycolysis via HIF-1α/PDK1 axis

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Protocol publication

[…] Briefly, 10×105 cells were seeded in 60 mm2 plates and cultured in the presence or absence of 5 μM 2-MeOE2 for 24 h. Following treatment, cells were lysed on ice with lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Samples were centrifuged at 12,000 × g for 10 min at 4°C, and supernatant was aliquoted and stored at −80°C for future use.Total protein (30 μg) was resolved in a 10% polyacrylamide gel using SDS-PAGE then transferred to a polyvinylidene difluoride (PVDF) membrane. Following transfer, membrane was blocked for 2 h at room temperature in 5% non-fat dry milk diluted in 0.1% Tween-20 in TBS (TBST) followed by an overnight incubation in blocking solution with primary antibodies. After washing, membranes were incubated for 2 h at room temperature with the appropriate peroxidase-conjugated secondary antibody, washed, and subjected to chemiluminescent substrate (Luminata Forte; Millipore Corp.). Membranes were imaged using the ChemiDoc XRS+ system (Bio-Rad, Hercules, CA, USA) and densitometry was performed using Image Lab software (Bio-Rad). Every target protein of our experiment was calculated by gray scanning using ImageJ (free software from NIH website), the relative density of target proteins were normalized to its marker internal protein. Primary antibodies used included rabbit anti-HIF1-α, β-actin, GLUT1, LDHA, PDK1. The quantification of band density was performed using ImageJ software. […]

Pipeline specifications

Software tools Image Lab, ImageJ
Application Microscopic phenotype analysis
Organisms Dipturus trachyderma, Homo sapiens
Diseases Melanoma, Neoplasms
Chemicals Adenosine Triphosphate, Pyruvic Acid, Lactic Acid