Apoptosis related to treatment with anticancer drugs. The number of apoptotic cells due to treatment with vinblastine (A), cisplatin (B) and cyclophosphamide (C) at IC50 doses in control conditions and after efflux pumps silencing in canine mammary cancer cell lines (CMT-U27, CMT-U309, P114, CMT-W1 and CMT-W2) assessed by the Annexin V/PI test (BD Bioscience, USA). All values were calculated versus control conditions (spontaneous apoptosis in these cell lines). The numbers of apoptotic cells are represented as a percentages of Annexin V-positive cells (obtained with FACS Aria II Becton Dickinson). The experiment has been conducted in three replicates. Error bars refer to S.D. p < 0.05 was marked as *, p < 0.01 was marked as ** and p < 0.001 was marked as ***. One-way ANOVA followed by Tukey HSD post-hoc test was applied. (D) The representative cytograms of P114 cell line double stained with Annexin V-FITC and propidium iodide (PI). The cytograms show P114 cells in control conditions, after cisplatin treatment at IC50 dose and after pgp, bcrp, mrp1 and mrp3 -specific siRNA treatment followed by cisplatin treatment at IC50 dose. On the cytograms are showed normal, early apoptotic, late apoptotic and necrotic cells. Left bottom quadrant shows normal cells, top left quadrant shows necrotic cells (stained with PI only; damaged cell membrane but no phosphatydilserine exposure), right bottom quadrant shows the early apoptotic cells (stained with Annexin V only; intact cell membrane) and top right quadrant shows cells in late stage of apoptosis (stained with Annexin V and PI - phosphatydilserine exposure and damaged cell membrane).