Ro5126766 ch5126766

Cells were seeded at a density of 2000 cells per well in 96 well plates. Twenty hours after seeding the medium was changed and 48 h after seeding (day 0) the cells were treated with 1 μM of DMSO or inhibitor(s) for three days. After completion of the drug exposure, cells were analyzed using the sulforhodamine B (SRB) colorimetric assay as described [38 (link)]. The relative cell growth was calculated on the average OD of the sample versus/normalized to average OD at day 0 and OD of the vehicle (DMSO)-treated control.
Inhibitors used in our study were: RO5126766 (CH5126766) (Selleckchem, Houston, TX, USA, S7170), GDC-0623 (Selleckchem, Houston, TX, USA, S7553), SCH772984 (Selleckchem, Houston, TX, USA, S7101). All inhibitors were dissolved in DMSO (D1435, Sigma Aldrich, St. Louis, MO, USA) and used at 1 μM final concentration. Final DMSO concentration was kept at 0.1% or below in control and inhibitor-treated cells for single treatments and between 0.2% and 0.3% in control and inhibitor-treated cells for double and triple treatments respectively.

Long-term drug resistance was assessed by seeding in parallel 2.5 × 10⁵ and 5 × 10⁵ cells in 6-cm plates and 24 h after plating cells were treated with 1 μM of a particular inhibitor as single or double treatment. For the plates where the initial cell number was 2.5 × 10⁵, medium and inhibitor(s) were changed every 3 days for 14 days. Cells were fixed and stained with Crystal Violet solution, pictures were captured with EPSON PERFECTION V300 PHOTO scanner and images acquired with the paired software. taken. For the plates whose initial cell number was 5 × 10⁵, medium and inhibitor(s) were changed every 3 days until plates were fully confluent. At confluence plates were split 1:5 to one 6-cm-plate without inhibitor(s) (1/5), in order to test for possible drug addiction, a 10-cm-plate with inhibitor(s) (3/5) for expansion/freezing and one 6-cm-plate with inhibitor(s) (1/5) for future protein isolation. Days after resistance were counted from the splitting time until the plates reached confluence again. Inhibitors used were RO5126766 (CH5126766) (Selleckchem, Houston, TX, USA, S7170), GDC-0623 (Selleckchem, Houston, TX, USA, S7553), SCH772984 (Selleckchem, Houston, TX, USA, S7101), LY3214996 (Selleckchem, Houston, TX, USA, S8534). Final DMSO concentration was kept at 0.1% in control and inhibitor-treated cells for single treatments and 0.2% for double treatments.