Computational protocol: Targeting protein homeostasis with nelfinavir/salinomycin dual therapy effectively induces death of mTORC1 hyperactive cells

Similar protocols

Protocol publication

[…] Tsc2−/− MEFs or ELT3-V3 cells were plated at 1000 cells/well into a 96-well plate, precoated with 1.5 % (w/v) agarose. Spheroids were allowed to form for 72 h before treatment with DMSO, 10 μM nelfinavir and 2 μM salinomycin or 25 nM rapamycin. Fresh media and drugs, including DRAQ7 (which is non-toxic), were added by removal and replacement of 50 % of the media volume after 60 h, and incubated for a further 36 h (total 96 h treatment incubation). Dual channel images were acquired using a Zeiss Axio Observer Z1 microscope (Carl Zeiss Microimaging, Gottingen, Germany) with a black box chamber (Solent Scientific Ltd, Segensworth, U.K.) at 0 and 96 h timepoints. Spheroid size (transmission mode) and DRAQ7 labelling (fluorescence excitation 488nm/emission above 695nm) were assessed using MetaMorph acquisition software. Following imaging, spheroids were transferred to a standard, tissue culture coated 24 well plate with fresh culture media (no drug treatments) and imaged using an EVOS XL Core camera (Life Technologies) after 0, 24, 48 and 72 h. Total outgrowth area from the spheroid was measured using ImageJ (v1.50i) software (https://imagej.nih.gov/ij/). […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Neoplasms, Tuberous Sclerosis, Drug-Related Side Effects and Adverse Reactions
Chemicals Cycloheximide, Nelfinavir, Sirolimus