Fig. 7: NCK synergizes with OSI-930 or Crizotinib to stimulate caspase-mediated apoptotic cell death and reduce cell survival in vitro and ex vivo.

a Representative flow cytometry plots using Annexin V FITC/PI staining for apoptotic cell death of MDA-MB-231 and BT549 cells measured after treatment with 5 μM NCK, 5 μM OSI-930 (OSI), 5 μM Crizotinib (CRI) or combinations using flow cytometry analysis at 72 hours as described in materials and methods (n = 3). b CASPASE 3/7 activities were evaluated in MDA-MB-231 and BT549 cells using the Biovision Caspase 3/7 DEVD Assay Kit after treatment with 5 μM NCK, 5 μM OSI-930 (OSI), 5 μM Crizotinib (CRI) or combinations. Data represent means ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. c Crystal violet staining of foci in colonies generated by MDA-MB-231 cells and BT549 cells after exposure to 5 μM NCK, 5 μM OSI-930 (OSI), 5 μM Crizotinib (CRI) or combinations. d Representative images of MDA-MB-231 cells and BT549 cells cultured in 3D Matrigel after exposure to 5 μM NCK, 5 μM OSI-930 (OSI), 5 μM Crizotinib (CRI) or combinations. Scale bar, 100μm. e Western blot analysis was used to assess the level of various apoptotic proteins in TNBC cells after treatment with 1 μM NCK, 1 μM OSI-930 (OSI), 1 μM Crizotinib (CRI) or combinations. β-ACTIN was used as input control for cell lysate. The sizes of detected bands in kDa are shown on the left. f Western blot analysis was used to assess the expression/phosphorylation of various proteins of the PI3K/AKT and MAPK pathways after treatment with 1 μM NCK, 1 μM OSI-930 (OSI), 1 μM Crizotinib (CRI) or combinations. β-ACTIN was used as input control for cell lysate. The sizes of detected bands in kDa are shown on the left.