FIGURE 4.

N134‐glycosylated GPNMB controls the oncogenic properties of mutated EGFR in vitro and in vivo. A, Lysates from different NSCLC cells with various EGFR status were treated with or without PNGase F, and both GPNMB and EGFR expressions were examined by immunoblotting (arrowhead: the unmodified GPNMB). B, Top: Localization of 11 glycosylation sites in the GPNMB gene structure. Bottom: Plasmids of 11 GPNMB glycosylated mutants were transfected into H1299 and EGFR downstream signaling was examined by immunoblotting. C, Effects of 11 GPNMB glycosylated mutants on cell migration by wound‐healing assay (n = 3 experiments, ** P < .01). D, Different EGFR mutants were co‐transfected with GPNMB‐wild‐type or N134Q plasmids into H1299 cells and immunoprecipitated with anti‐FLAG antibodies to detect the effects on the binding affinities between GPNMB‐wild‐type/N134Q and various EGFR mutants (arrowheads: the precipitated GPNMB quantified under the blot). E, H1299 cells were transfected with the indicated EGFR and GPNMB mutant plasmids, and the effects on cell migratory and invasive abilities were examined (*P < .05 and ** P < .001). Protein expression of transfected EGFR and GPNMB are shown on the left. F, Tail‐vein metastatic assay in vivo. Left: the growth (top) and H&E staining (bottom) of lung tissues from each group. Right: number of tumor nodules of each group (** P < .01)