Accuri c6 analysis

Cell cycle analysis by FACS was performed as recently described [43] , using propidium iodide (PI) staining and flow cytometry (BD Accuri C6 Analysis). GOT1 cells (3 × 10 5 cells/well) were cultured for 24 h in complete medium. The next day, the medium was replaced with fresh 10% FBS medium and incubated with NVP-CGM097 (1-3 μ M ), 5-fluorouracil (10 μ M ), and octreotide (10 μ M ). After 72 h, the cells were washed with PBS and treated with 300 μL trypsin at 37 ° C for 4 min. The cells were collected and centrifuged at 2,000 rpm for 5 min. After another wash cycle with PBS, the cells were centrifuged again. The pellets were resuspended in 350 μL PI. After 2 h, the samples were measured.
Caspase Activity Assay GOT1 cells (1 × 10 5 cells/well) were incubated with NVP-CGM097 (1-3 μ M ), 5-fluorouracil (10 μ M ), and octreotide (10 μ M ) for 72 h. Afterwards, caspase activity was measured with the APO-One ® homogenous caspase 3/7 assay (Promega, Mannheim, Germany) following the manufacturer's instructions.

Cell cycle distribution was analyzed following the quick method from Nature Protocols "Analysis of apoptosis by propidium iodide staining and flow cytometry" [42] (BD Accuri C6 Analysis). Cells were cultured in 6-well plates for 24 h, and then the medium was replaced by fresh and antibiotics-free medium, and cells were incubated with different concentrations of AR-A014418 alone (5-60 µM) or in combination with lovastatin (20 µM AR-A014418 + 10 µM lovastatin). After 48 h, cells were washed with PBS and treated with 300 μL trypsin for 5 min at 37 ° C. The cells were collected, washed, and resuspended in 300 μL propidium iodide (Sigma-Aldrich), and 8 h later 20,000 events from each sample were analyzed.

Cell cycle phase distribution was analyzed using propidium iodide staining and flow cytometry (BD Accuri C6 Analysis, BD Biosciences). Cells were cultured in 6-well plates for 24 h. Subsequently, medium was replaced by fresh medium and cells were incubated with different concentrations of GNF-5837. After 72 h, cells were washed with PBS and treated with 300 μL trypsin for 5 min at 37°C. Cells were collected, washed and resuspended in 300 μL propidium iodide solution (Sigma-Aldrich).

Cell cycle distribution was analyzed using propidium iodide staining and flow cytometry (BD Accuri C6 Analysis). Cells were cultured in 6-well plates (4 × 10 5 BON1 cells/well and 5 × 10 5 NCI-H727 cells/well) for 24 h in complete medium. The next day, the medium was replaced with fresh 10% FBS medium and incubated with 10 μ M INC280, cabozantinib and tivantinib. After 24, 48 and 72 h, cells were washed with PBS and treated with 300 μl trypsin at 37 ° C for 4 min. Cells were collected and centrifuged at 2,000 rpm for 5 min. After another wash cycle with PBS, the cells were centrifuged again. The pellets were resuspended in 350 μl propidium iodide. After 2 h, the samples were measured.