Computational protocol: Mechanisms of the anti-tumor activity of Methyl 2-(-5-fluoro-2-hydroxyphenyl)-1 H-benzo[d]imidazole-5-carboxylate against breast cancer in vitro and in vivo

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Protocol publication

[…] MCF-7 and MDA-MB-231 cells were seeded onto 22 × 22 mm cover slips in a 6-well plate, and subsequently were treated with different concentrations of MBIC. After 24 h, cells were fixed with 4% paraformaldehyde and stained using Cellomics Multiparameter Cytotoxicity 3 Kit (#8408102) according to the manufacturer's protocol. This kit includes dyes that represent morphological changes in the nucleus (blue at 405 nm; channel 1), cell membrane permeability (green at 488 nm; channel 2), MMP (red at 543 nm; channel 3) and release of cytochrome c (cyan at 633 nm; channel 4). Cells were incubated with rabbit anti-mouse DyLight 649-conjugated secondary antibody and Hoechst 33342 (Thermo Fisher Scientific, USA). All confocal fluorescence images were acquired using LAS AF software, version 2.6.0.7266 in Leica TCS SP5 II confocal system (Mannheim, Germany) with a HCX PL APO LAMBDA BLUE 40 ×/1.25 oil objective. Data were processed using LAS X software, version 3.0.0. Each experiment was performed in triplicate. [...] Cells were treated with MBIC in a dose-dependent manner for 24 h. Cells were harvested, lysed and applied for Western blot analysis as described previously []. The primary antibodies that were used in this study included: anti-p53, anti-survivin, anti-phospho-survivin (anti-p-survivin), anti-Cdk1, anti-cyclin B1, anti-Bax, anti-caspase-3, anti-caspase-7, anti-caspase-9 (1:1000) (Cell Signaling Technology, USA) and mouse anti-β-actin (1:40000) (Sigma-Aldrich, St. Louis, MO, USA) antibodies. Western blot images were quantified and processed by ImageJ software (NIH, USA). Each experiment was done in triplicate. […]

Pipeline specifications

Software tools LAS X, ImageJ
Application Microscopic phenotype analysis
Organisms Mus musculus
Diseases Breast Neoplasms, Heart Arrest, Neoplasms, Drug-Related Side Effects and Adverse Reactions