Prolong antifade 4 6 diamidino 2 phenylindole mounting medium

SW15743 cells were seeded (50,000 cells per well) on each chamber of the Lab-Tek II chamber slide system (four-well format, catalog no. 12-565-7, Thermo Fisher Scientific). After 12 hours of cell seeding, the cell medium of each chamber was replaced with the fresh medium for the untreated chamber and adagrasib medium for the treated chambers (0.5, 1, or 2 μM). The cells were allowed to grow for 72 hours, followed by fixation with 4% formaldehyde for 30 min at room temperature, and blocked using the blocking buffer (catalog no. 12411, Cell Signaling Technology). Primary antibody against WNT2 (1:400 dilution; catalog no. 66656-1-Ig, Proteintech, USA) and phospho-S675–β-catenin (1:100 dilution; catalog no. 4176, Cell Signaling Technology, USA) were used for overnight staining at 4°C. The slides were washed, and secondary antibody was incubated for 2 hours followed by washing at room temperature. ProLong Antifade 4′,6-diamidino-2-phenylindole mounting medium (catalog no. 8961, Cell Signaling Technology, USA) was used for mounting the coverslips and imaged with Zeiss LSM 880 confocal microscope at the Light Microscopy/Digital Imaging Core Facility at City of Hope.

SW15743 cells were seeded (50,000 cells per well) on each chamber of the Lab-Tek II chamber slide system (four-well format, catalog no. 12-565-7, Thermo Fisher Scientific). After 12 hours of cell seeding, the cell medium of each chamber was replaced with the fresh medium for the untreated chamber and adagrasib medium for the treated chambers (0.5, 1, or 2 μM). The cells were allowed to grow for 72 hours, followed by fixation with 4% formaldehyde for 30 min at room temperature, and blocked using the blocking buffer (catalog no. 12411, Cell Signaling Technology). Primary antibody against WNT2 (1:400 dilution; catalog no. 66656-1-Ig, Proteintech, USA) and phospho-S675–β-catenin (1:100 dilution; catalog no. 4176, Cell Signaling Technology, USA) were used for overnight staining at 4°C. The slides were washed, and secondary antibody was incubated for 2 hours followed by washing at room temperature. ProLong Antifade 4′,6-diamidino-2-phenylindole mounting medium (catalog no. 8961, Cell Signaling Technology, USA) was used for mounting the coverslips and imaged with Zeiss LSM 880 confocal microscope at the Light Microscopy/Digital Imaging Core Facility at City of Hope.