60 mm cell culture dishes

A375 pLNCX2^empty, A375 pLNCX2-PIK3CA^wt, and A375 pLNCX2-PIK3CA^H1047R were seeded in 60 mm cell culture dishes (Thermo Fisher Scientific, Waltham, MA, USA) at a density of 3 × 10⁵ cells. After 24 h, each infected cell line was treated with increasing concentrations of the selected drugs: dabrafenib (0.0–0.5–2.0 nM), BYL719 (0.0–0.25–1.0 μM), and trametinib (0.0–0.5–1.0 nM). Combined treatments were obtained as follows: trametinib (0.0–0.25–0.5 nM) and dabrafenib (1.0 nM) for each trametinib dilution; BYL719 (0.0–0.25–1.0 μM) and dabrafenib (1.0 nM); and BYL719 (0.0–0.25–1.0 μM) and trametinib (0.5 nM). DMSO was used as control. All cell lines were treated for 48 h in standard culture conditions.
For each treatment, adherent cells were collected by scraping. Cell pellets were collected by centrifugation and stored at −80 °C. All experiments were conducted in triplicate.
Fixed doses of dabrafenib or trametinib in combined treatments (BYL719/Dabrafenib and BYL719/Trametinib, respectively) were used to evaluate the inhibitory responsiveness of MAPK pathway under gradual modulation of Akt signalling through different doses of BYL719. Similarly, increasing doses of trametinib were adopted in Trametinib/Dabrafenib treatment to investigate the effect of downstream inhibition of MAPK signalling on the cellular response to dabrafenib.

Cells were seeded into 60-mm cell culture dishes (Thermo Fisher Scientific) to achieve 30–40% confluency. The next day, the cells were treated with drugs as described above. After treatment, cells were harvested by mild trypsinization, resuspended in 300 μL of ice-cold PBS, and fixed by the addition of 0.7 mL of ice-cold 70% ethanol in a dropwise manner with constant mixing. After addition of ethanol, samples were stored at −70 °C overnight. Fixed cell samples were washed with ice-cold ethanol twice and stained with 1 µg/mL DAPI (R&D Systems). Stained samples were analyzed using CytoFLEX flow cytometer and CytExpert software (Beckman Coulter), at least 25 000 qualifying events were detected in each evaluated sample.