Cell culture. Panc-1 and HEK293 Z-VDVAD-FMK were grown in Minimal Essential Medium (Invitrogen, Carlsbad, CA), whereas MCF-7 and A549 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Nisssui, Tokyo). Both media were supplemented with 5% FBS and 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA). Cell culture was carried out at 37 °C and 5% CO2.
Treatment with antibodies. To determine the internalization of MUC1, the cells were treated for 1 h on ice with 4 μg/ml of anti-MUC1 antibody in the culture medium containing 5% FBS, washed twice with ice-cold PBS, and incubated at 37 °C for 1 h in fresh culture medium. In the experiments dealing with signaling, the cells were treated with 4 μg/ml of antibodies at 37 °C for various time periods in the culture medium containing 5% FBS. At the end of the treatment, the cells were washed twice with ice-cold PBS and treated with methanol at ?20 °C for 20 min for fixation and permeabilization followed by blocking with 1% BSA in PBS for 20 min at room temperature and staining with TRITC-conjugated goat secondary antibody against mouse IgG. Fluorescent signals were visualized by confocal laser microscopy (FLUOVIEW FV300, Olympus, Japan).