Cell viability was determined at the same time of treatment and after 48 h. To determine the number of viable cells, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well and incubated during 3 h, subsequently the medium was removed and 200 μL of DMSO (dimethyl sulfoxide) was added in order to lyse the SB431542 and resuspend the formazan (MTT metabolic product). Quantities of formazan product, which is directly related to the number of viable cells, were measured at 560 nm using a scanning spectrophometer microplate reader (UVM 340 Biochrom, Cambridge). Concentration values corresponding to the parameters IC50 (50% cell viability inhibition), GI50 (50% growth inhibition), TGI (total growth inhibition) and LC50 (50% cell death) were calculated according to the NIH definitions using a logistic regression.
2.3.3. Preparation of mixed micellar and control solution
Micelles were prepared with egg yolk lecithin and sodium taurocholate at final concentrations of 150 μM and 0.5 mM respectively. Micelle solutions were prepared by first adding appropriate amounts of each treatment compounds to a glass vial containing 0.5 mL of hexane. For control solutions, a vial with 0.5 mL of hexane was prepared. Vials content were dried using a nitrogen evaporator (N-Evap 111, Organomation Associates). Then 0.23 mg of lecithin dissolved in 0.575 mL of hexane was added to each vial and dried again in the same conditions; this was followed by the addition of 500 μL of 2 mM taurocholate prepared in ethanol and another evaporation step. Finally, dried content was resuspended in 1 mL of DMEM without supplements and mixed with 1 mL of complete DMEM, and 200ul of this mixture was added to each well.