Role of ANKHD1 in NSCLC cell proliferation and invasion; *P<0.05. (A) Colony formation assay demonstrated that ANKHD1 overexpression in A549 cells promoted colony formation, whereas ANKHD1 depletion inhibited colony formation in LK2 cells. (B) Transwell assay demonstrated that ANKHD1 over-expression in A549 cells promoted invasion, whereas ANKHD1 depletion inhibited invasion in LK2 cells (magnification, x100; scale bar, 100 µm). (C) MTT assay also demonstrated that ANKHD1 overexpression in A549 cells increased proliferation, whereas ANKHD1 depletion decreased proliferation in LK2 cells. (D) ANKHD1 regulated NSCLC growth in vivo. Mice that received A549 cells stably expressing ANKHD1 (bottom row, G418 screening) exhibited an increase in tumor weight compared with the control group (upper row), whereas animals that received LK2 cells transduced with lentiviral shRNA-ANKHD1 (upper row, G418 screening) exhibited a reduction in tumor weight compared with the control group (bottom row). (E) ANKHD1-expressing A549 cells exhibited more progressive tumor growth in the nude mice (n=3) compared with the control group (n=3). Consistently, the ANKHD1-depleted LK2 cell inoculation resulted in lower proliferative ability in the nude mice (n=3) compared with the control group (n=3). (F) Transfection of plasmid pCMV6-Myc/DDK-ANKHD1 for ANKHD1 overexpression in A549 cells, compared with negative plasmid. siRNA-ANKHD1 to knockdown the expression of ANKHD1 in LK2 cells, compared with negative plasmid. The transfection efficiency was determined by western blotting. ANKHD1, ankyrin repeat and KH domain-containing 1; NSCLC, non-small-cell lung cancer.