A375 cells were cultured in 35 mm glass bottom dishes (MatTek), incubated with fluorescein-conjugated cholera toxin B subunit (FITC-CTXb; Sigma) at a concentration of 10 μg/ml in D-PBS for 30 min at 37 °C. Cells were then washed with D-PBS and treated with 1 μM AZD6244 in complete RPMI-1640 medium for 24 h.
For confocal fluorescence microscopy analysis, cells were fixed with 4 % PFA, permeabilized with 0.25 % Triton X-100, then stained with appropriate primary antibodies followed by Alexa Fluor antibodies (Life Technologies) as secondary antibodies. Counter staining of cell nuclei was performed using DAPI (Santa Cruz Biotechnology). Immunofluorescence images were acquired with Zeiss LSM880 Airyscan confocal laser scanning microscope (Zeiss) with ×63 glycerol-immersion objective and scanning resolution of 512 × 512 pixels, zoom factor 6.4 for a subset of images. Immunofluorescence intensity were analyzed using ImageJ Software (NIH,version 1.52).
For Total internal reflection fluorescence structured illumination microscopy (TIRF-SIM) analysis, cells were fixed with 4 % PFA, and mounted in ProLong™ Gold Antifade Mountant (Thermo Fisher). Immunofluorescence images were acquired with DeltaVision OMX SR imaging system (Cytiva).
For confocal fluorescence microscopy analysis, cells were fixed with 4 % PFA, permeabilized with 0.25 % Triton X-100, then stained with appropriate primary antibodies followed by Alexa Fluor antibodies (Life Technologies) as secondary antibodies. Counter staining of cell nuclei was performed using DAPI (Santa Cruz Biotechnology). Immunofluorescence images were acquired with Zeiss LSM880 Airyscan confocal laser scanning microscope (Zeiss) with ×63 glycerol-immersion objective and scanning resolution of 512 × 512 pixels, zoom factor 6.4 for a subset of images. Immunofluorescence intensity were analyzed using ImageJ Software (NIH,version 1.52).
For Total internal reflection fluorescence structured illumination microscopy (TIRF-SIM) analysis, cells were fixed with 4 % PFA, and mounted in ProLong™ Gold Antifade Mountant (Thermo Fisher). Immunofluorescence images were acquired with DeltaVision OMX SR imaging system (Cytiva).