Selective transfer of P-gp to MCF-7 breast cancer cells by DXMPs by Western Blot analysis.

<p>20 µg total cell lysates of non-malignant cells (breast epithelial cells (MBE), osteoblasts (HO-f) and urothelial (HUC) cells) and their co-cultures; and drug sensitive cancer cells (leukaemia, CEM and breast cancer, MCF-7) and their co-cultures with leukaemic cell-derived (<b>A</b>) VLBMP and (<b>B</b>) E₁₀₀₀MP overexpressing P-gp and MRP1, respectively, and breast cancer-derived (<b>C</b>) DXMP overexpressing P-gp, were analyzed by Western blot analysis. (<b>A</b>) P-gp and (<b>B</b>) MRP1 were readily detected in the drug resistant leukaemic donor cells, their MPs and also in both the recipient malignant and non-malignant cells after MP transfer using the anti-P-gp mAb, F4 and anti-MRP1 mAb, QCRL-1, respectively. (<b>C</b>) P-gp was also detected in the drug resistant breast cancer donor cell, its MPs (DXMP), recipient malignant drug sensitive cells but not in the recipient non-malignant cells after MP transfer. α-tubulin or β-actin were used as internal loading controls. (<b>D</b>) <b>Selective transfer of P-gp to MCF-7 breast cancer cells by malignant breast cancer-derived MPs by Confocal microscopy.</b> Malignant breast cells MCF-7 (<b>I</b>) or non-malignant osteoblasts, HO-f (<b>II</b>) cells were stained with fluorescent membrane probes PKH26 and the nucleus was stained with DAPI after 4 hours co-culture with GFP-MPs. (<b>I</b>) P-gp was detected in the breast MCF-7 cells only and not in (<b>II</b>) the osteoblasts. Panel A shows all channels captured, Panel B shows the DAPI in the 405 nm channel, Panel C shows the transferred GFP (or lack of) in the 488 nm channel, and Panel D shows the PKH26 in the 561 nm channel. Images were acquired using the Nikon A1 confocal microscope. Scale bar as indicated. Both Western blot and confocal microscopy experiments were repeated at least 2–3 times with similar results. Data are representative of a typical experiment.