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Cells were maintained in a humidified environment of 5% CO 2 at 37 C, cultured until an optical confluence of 75-100%, detached with 0.05% trypsin-EDTA 1 (25300-054, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA), at 37 C for approximately 5 min, and split to new plates according to the experimental procedures