Computational protocol: Bis-anthracycline WP760 abrogates melanoma cell growth by transcription inhibition, p53 activation and IGF1R downregulation

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Protocol publication

[…] Cells were trypsinized, washed with PBS−, and lysed in IP or RIPAS buffer supplemented with protease inhibitor cocktail (Roche). The lysates were incubated for 15 min on ice, centrifuged, and frozen. Immunoblotting was performed using aliquots (15–30 μg) of the whole-cell extract. Proteins were separated on polyacrylamide gels (8 or 12%) and blotted onto PVDF (Immobilon-P Transfer Membrane; Millipore) or nitrocellulose (Amersham Protran Premium) membranes. Antibodies used for detection are listed in Supplementary material (Table ). The chemiluminescent substrate signal (Thermo Scientific; 34079, 34095) was developed using the Curix60 processor (Agfa). [...] Global RNA synthesis was evaluated using Click-it RNA imaging kit (Thermo Fisher Scientific, C10330). Cells were grown on glass coverslips for 24 h in complete culture medium containing WP760 (10 nM, 50 nM, and 100 nM), or 25 nM actinomycin D, (Sigma-Aldrich). Briefly, staining was performed as follows: ethynyl uridine (EU) was added to the culture medium 1 h before the end of incubation with the tested compounds. Cells were then rinsed, fixed in 3.7% formaldehyde, and washed with PBS followed by staining with 10 μM Alexa594-azide. Subsequently, cells were washed with PBST, counterstained with Hoechst 33342, and visualized by confocal microscopy (Zeiss LSM750). Signal intensity was evaluated using ImageJ software. […]

Pipeline specifications

Software tools PReMiuM, ImageJ
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Melanoma, Neoplasms, Drug-Related Side Effects and Adverse Reactions