Fig 2. TK1 inhibition reduces growth and metastasis of LUAD cell lines.

(A) TK1 levels were measured by immunoblot analysis in LUAD cell lines expressing TK1 shRNAs or control, NS shRNA. ACTINB was used as a loading control. (B) (Left) Anchorage-independent growth was measured by soft-agar assay in LUAD cell lines expressing either TK1 short hairpin RNA (shRNA) or a non-specific (NS) shRNA control. Representative images of soft-agar colonies from the indicated LUAD cell lines are shown. Scale bar, 500 μm. (Right) Plot showing relative colony sizes from the soft-agar assay presented in panel on left. (C) (Left) Matrigel invasion assays with the indicated LUAD cell lines expressing TK1 shRNA or NS shRNA; representative images are shown. Scale bar, 200 μm. (Right) Relative invasion (%) from Matrigel assays shown in the left panel. (D) (Left) Wound-healing assays with LUAD cells expressing TK1 shRNA or NS shRNA control. Representative images at the indicated times are shown. Scale bar, 200 μm. (Right) Relative migration (%) calculated from the data presented on the left. (E) LUAD cell lines expressing either TK1 shRNA or NS shRNA were subcutaneously injected into the flanks of athymic nude mice (n = 3). Average tumor volumes at the indicated time points are shown. (F) TK1 levels were measured by immunoblot analysis in A549-F-Luc cells expressing TK1 shRNAs or NS shRNA. ACTINB was used as a loading control. (G) A549-F-Luc cells expressing TK1 shRNA or NS shRNA were administered to NSG mice (n = 5) via tail vein injection. Bioluminescence images of mice from the indicated groups at weeks 1 and 5 are shown. (H) (Left) Representative images of hematoxylin and eosin (H&E)-stained lung sections from the week 5 groups shown in panel G. (Right) Relative size of metastatic nodules in week 5 lungs with A549-F-Luc cells expressing TK1 shRNA or NS shRNA. Data are presented as the mean ± SEM; *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively.