FIGURE 1.

Identification of EMGs in LUAD. (A) A volcano plot indicating upregulated (red dots) and downregulated (green dots) genes in the LUAD tissues of patients with bone metastasis compared with those without bone metastasis. Black dotted lines represent a cut‐off range of 2.0‐fold and p < .01. (B) A volcano plot showing 4 upregulated (red dots) genes associated with endocytosis in the LUAD tissues of the patients with bone metastasis compared with those without bone metastasis. Black dotted lines represent a cut‐off range of 2.0‐fold and p < .01. VPS37A, EHD1, GRK5 and VPS4A were identified as the candidate EMGs. (C) Kaplan–Meier curves for the overall survival of patients with LUAD according to the expression levels of the candidate EMGs. HR, hazard ratio; CI, confidence interval. (D and E) The effects of EHD1 knockdown in A549 cell lines were examined by qRT‐PCR and Western blot analysis. (F–H) The effect of EHD1 depletion on the stemness of A549 cells was determined in vitro. (F) 3D spheroid assays using semisolid medium, (G) 3D spheroid assays using serum‐free medium and (H) holoclone assays showed the CSCs‐like traits of A549 cells. (I) The expression of stemness‐related markers in A549 cells. (J) Flow cytometric analysis of CD133‐ and CD44‐positive cells. (K) In the in vitro limiting dilution assays, the numbers of wells in 96‐well plates that contained tumour spheres were determined (upper panel). The stemness of A549 cells with or without EHD1 knockdown was estimated as the stem cell frequency (bottom panel). The data are shown as the mean ± standard deviation (SD) values. p > .05 was considered not significant (N.S.), *p < .05, **p < .01 and ***p < .001