Lentivirus-mediated knockdown of NLK inhibits small-cell lung cancer growth and metastasis

Figures & data

Figure 1 Expression patterns of NLK in SCLC and adjacent lung tissues.

Note: qRT-PCR analysis of NLK expression in 20 paired SCLC and adjacent lung tissue samples.
Abbreviations: NLK, Nemo-like kinase; qRT-PCR, quantitative real-time polymerase chain reaction; SCLC, small-cell lung cancer.

Figure 2 Lentivirus-delivered shRNA targeting NLK downregulated its endogenous expression.

Notes: (A) GFP expression in the uninfected and lentivirus-infected NCI-H446 cell lines recorded by fluorescence microscopy. (B) qRT-PCR analysis of NLK knockdown efficiency in the uninfected and lentivirus-infected NCI-H446 cells. (C) Representative immunoblot showing NLK knockdown efficiency determined in the uninfected and lentivirus-infected NCI-H446 cells. The β-actin gene and protein were used as internal controls. Data are mean ± SD (n=3; t-test). *P<0.05.
Abbreviations: Con, control; GFP, green fluorescence protein; Lv-shCon, lentivirus-containing non-silencing shRNA; Lv-shNLK, lentivirus-containing NLK shRNA; NLK, Nemo-like kinase; qRT-PCR, quantitative real-time polymerase chain reaction; shRNA, short hairpin RNA.

Figure 3 Depletion of NLK decreased cell proliferation and colony formation.

Notes: (A) Growth curves of the uninfected and lentivirus-infected NCI-H446 cell lines determined by CCK-8 assay. (B) Representative colony formation showing clonogenic survival determined in the uninfected and lentivirus-infected NCI-H446 cells. (C) Statistical analysis of colony numbers in the uninfected and lentivirus-infected NCI-H446 cells. Data are mean ± SD (n=3; t-test). *P<0.05.
Abbreviations: CCK-8, Cell Counting Kit-8; Con, control; Lv-shCon, lentivirus-containing non-silencing shRNA; Lv-shNLK, lentivirus-containing NLK shRNA; NLK, Nemo-like kinase; shRNA, short hairpin RNA.

Figure 4 Downregulation of NLK induced S-phase cell cycle arrest.

Notes: (A) Flow cytometric analysis of cell cycle distributions in the uninfected and lentivirus-infected NCI-H446 cells. (B) Statistical analysis of G1-, S-, G2/M-phase populations in the uninfected and lentivirus-infected NCI-H446 cells. Data are mean ± SD (n=3; t-test). *P<0.05; **P<0.01.
Abbreviations: Con, control; Lv-shCon, lentivirus-containing non-silencing shRNA; Lv-shNLK, lentivirus-containing NLK shRNA; NLK, Nemo-like kinase; shRNA, short hairpin RNA.

Figure 5 Downregulation of NLK impeded cell migration.

Notes: (A) Representative crystal violet staining showing cell migration determined in the uninfected and lentivirus-infected NCI-H446 cells. (B) Statistical analysis of migrated cells in the uninfected and lentivirus-infected NCI-H446 cells. Data are mean ± SD (n=3; t-test). *P<0.05.
Abbreviations: Con, control; Lv-shCon, lentivirus-containing non-silencing shRNA; Lv-shNLK, lentivirus-containing NLK shRNA; NLK, Nemo-like kinase; shRNA, short hairpin RNA.

Figure 6 Downregulation of NLK affected cell cycle and migration regulatory proteins.

Notes: Representative immunoblot showing expression alterations of Cyclin A, CDK2, CDC25A, and MMP-9 determined in the uninfected and lentivirus-infected NCI-H446 cells. β-actin protein was used as an internal control.
Abbreviations: Con, control; Lv-shCon, lentivirus-containing non-silencing shRNA; Lv-shNLK, lentivirus-containing NLK shRNA; MMP-9, matrix metalloproteinase 9; NLK, Nemo-like kinase; shRNA, short hairpin RNA.

Figure 7 Downregulation of NLK inhibited tumor growth and lung metastasis in vivo.

Notes: (A) Growth curves of xenograft tumors in nude mice treated with lentivirus-infected NCI-H446 cells. (B) Statistical analysis of weights of xenograft tumors in nude mice treated with lentivirus-infected NCI-H446 cells. (C) Representative images recorded under light microscope, representing the size of tumors in nude mice. (D) The tumor was confirmed by H&E staining. (E) Incidence of tumor lung metastasis in nude mice induced by injection of NCI-H446 cells, which were stably infected with Lv-shNLK or Lv-shCon. (F) Lung metastases in the mice were confirmed by H&E staining; magnification ×100. Data are mean ± SD (n=3; t-test). *P<0.05; **P<0.01.
Abbreviations: Con, control; Lv-shCon, lentivirus-containing non-silencing shRNA; H&E, hematoxylin and eosin; Lv-shNLK, lentivirus-containing NLK shRNA; NLK, Nemo-like kinase; shRNA, short hairpin RNA.