Figure 1: Ctx^R clones and xenografts have increased abundance of nEGFR and AXL.

(A) Immunoblotting (top, left) in non-nuclear and nuclear lysates harvested from three Ctx^R clones (HC1, HC4, and HC8) and the Ctx^S parental cell line HP. Calnexin, α-Tubulin, and histone H3 were used as loading and purity controls for non-nuclear and nuclear lysates, respectively. Structured resolution microscopy (bottom) in parental (HP) and Ctx^R clones stained for DAPI (blue) and EGFR (red) (bottom). Magnification, 100X. Scale bars, 10μm. Transmission electron microscopy (TEM; right) of fixed HC4 cells labeled with EGFR antibody-bound gold particles. Black arrows in insets mark gold particles in the nucleus. Cyto, cytoplasm; NE, nuclear envelope; nPore, nuclear pore; Nuc, nucleus. Scale bars, 0.5 μm. (B) Immunobloting in whole cell lysates from HP and Ctx^R clones. GAPDH: loading control. (C) Tumor volume (left) and immunohistochemical staining for EGFR and AXL in representative NCI-H226 xenografts (right) from IgG- or cetuximab-treated mice (n=4 and 5 mice, respectively; representative sections from 3 of each group are shown). Magnification, 40X. Scale bars, 1000 μm. (D) Immunohistochemical staining for EGFR and AXL abundance in Ctx^S and Ctx^R NSCLC PDXs. Magnification, 20X. Scale bars, 1000 μm. Black arrows in insets mark nEGFR. nEGFR and AXL abundance were quantified with ImageJ software. AXL staining intensity in Ctx^R tumors was normalized to that of the IgG or Ctx^S tumors (n=6 mice; representative sections from 3 of each group are shown). Data are means ± SD of 3 independent fields of view per tumor. * P < 0.05; ** P < 0.01, by two-tailed Student t-test. Blots and microscopy are representative of 3 experiments.