Influence of trastuzumab and T-DM1 on cell cycle phase distribution and cell signalling. (A) Calu-3 cells were cultured in the absence of drugs or treated either with T-DM1 or trastuzumab (1 μg/ml). After 24 h cells were stained with propidium iodide an cell-cycle-phase distribution was determined by flow cytometry analysis. Cell-cycle distributions were analyzed as described in Methods section and data were expressed as percentage of distribution in each cell-cycle phase. Immunoblot analysis on protein involved in cell cycle regulation (B) or signalling pathways (C) were conducted on cell lysates obtained after treatment with trastuzumab or T-DM1 (1 μg/ml) for the indicated period of time.