Fig. 5

Mechanism of AMD3100 function in HER2 + breast cancer with trastuzumab resistance. A BTRT cells grown in 3D Matrigel culture were treated with vehicle, AMD3100 (10 µM), trastuzumab (20 µg/ml), or their combination. SDF-1α (4 ng/ml) was added at the same time. Cell lysates were subject to RPPA (“Materials and methods” section). Data are presented in a matrix format: each row represents an antibody target and each column a sample. In each sample, the ratio of the abundance of the molecule to its median abundance across all samples is represented by the color of the corresponding cell in the matrix (see the scale for expression levels). B The cells were treated the same as A. Cell lysates were subjected to western blot analysis. C BTRT and SKRT cells grown in 3D Matrigel culture were treated with AMD3100 or vehicle for 3 days. SDF-1α (4 ng/ml) was added at the same time. The cells were collected from the Matrigel and analyzed to determine the phases of the cell cycle using flow cytometry. D, E Quantitative analysis was performed, and the data were analyzed using one-way ANOVA. The data are reported as mean ± SD of triplicates, representing two independent experiments (*P < 0.001, **P < 0.0001 compared with vehicle). F SKRT cells grown on coverslips pre-coated with poly-l-lysine were treated with AMD3100 or vehicle in the growth medium for 72 h. SDF-1α (4 ng/ml) was added at the same time. The cells were stained with the Kwik Diff Stains kit (scale bar, 50 μm). G SKRT cells were similarly grown on coverslips and treated with AMD3100 or vehicle in the growth medium for 48 h. After serum starvation overnight, the cells received SDF-1α (100 ng/ml) stimulation for the times indicated and were fixed and permeabilized. CXCR4 was detected with mouse antihuman CXCR4 primary antibody (R&D, Minneapolis, MN) and the Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (green). Nuclei were stained with DAPI (blue). Microscopic images were captured by a multiphoton confocal laser scanning microscope (“Materials and methods” section). Arrows indicate CXCR4 nuclear translocation, white triangles indicate binucleated cells, and yellow triangles indicate giant multinucleated cells