Labssystems multiskan ms microplate reader

For determining cell viability and proliferation, compound 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used as previously described [20 (link)]. Cells were treated with different HH-GLI pathway inhibitors using the concentration ranges that correspond to previously published studies: GANT61 5–25 μM (Selleck Chemicals, Houston, TX, USA), cyclopamine (CYC) 1.25–10 µM (Selleck Chemicals, Houston, TX, USA), and lithium chloride (LiCl) 5–40 mM (Kemika, Zagreb, Croatia) for 24–72 h [21 (link),22 (link),23 (link)]. Cell viability was measured on LabsSystems Multiskan MS microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 570 nm. The treatment was performed in quadruplicate for each dose, and the experiment was repeated twice.

In order to determine cell viability, compound 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used as previously described [12 (link)]. Cells were plated in 96-well plates at 2 × 10³ cells/well. At 24 h post-plating, cells were treated with HH-GLI and RAS/RAF/MAPK pathway inhibitors—GANT-61 2.5–30 μM (GLI antagonist, Selleck Chemicals, Houston, TX, USA); cyclopamine (CYC, SMO antagonist) 1.25–10 nM (Selleck Chemicals, Houston, TX, USA); vismodegib (VDG, SMO antagonist) 1–50 μM (Selleck Chemicals, Houston, TX, USA); sonidegib (SDG, SMO antagonist) 1–50 μM (Selleck Chemicals, Houston, TX, USA); lithium chloride (LiCl, GSK3ß antagonist) 1–40 mM (Kemika, Zagreb, Croatia); arsenic trioxide (ATO, GLI antagonist) 0.03–125 μM (Sigma-Aldrich, St. Louis, MI, USA, SAD); and salirasib (SAL, RAS antagonist) 1.6–200 μM (Sigma-Aldrich, St. Louis, MI, USA, SAD)—for 72 h. Absorbance was measured on the LabsSystems Multiskan MS microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 570 nm. The treatment was carried out in quadruplicate for each dose, and the experiment was repeated twice.