Figure 2

ALKBH4 knockdown suppressed NSCLC cells proliferation. Lysates of A549 (A) and II-18 (B) cells transfected with two ALKBH4 siRNAs (#1 and #2) or control siRNA, A549 cells transfected with pEB Multi-Neo-ALKBH4 or mock vector (C) were subjected to Western blot analysis with anti-ALKBH4 and anti-β-actin antibodies. Uncropped Western blot data are shown in Supplementary Fig. S9. Representative pictures of three independent experiments are shown (upper pictures). The cells which were transfected for 48 h were reseeded on a xCELLigence E-plate and their proliferation was detected using a xCELLigence DP system (lower panels). Degrees of cell proliferation expressed as cell index by the system are the means ± S.D. of three independent experiments (A–C). (D) Lysates of NCI-H23 cells co-transfected with pEB Multi-Neo-ALKBH4 wild-type (WT), pEB Multi-Neo-ALKBH4 mutant (mt), or mock vector and ALKBH4 siRNAs were subjected to western blot analysis with anti-ALKBH4 and anti-β-actin antibodies. Uncropped Western blot data are shown in Supplementary Fig. S9. Representative pictures of three independent experiments are shown (upper pictures). NCI-H23 cells were transfected for 48 h and reseeded in 96-well plates, and their proliferation was measured using the WST-8 assay (lower panels). The relative cell growth data on day 4 are presented in (E). The values are presented as the mean ± S.D. for each group. *p < 0.05; **p < 0.01 vs. wild-type ALKBH4 (one-way ANOVA with Bonferroni post-hoc tests). (F) A549 cells stably expressing ALKBH4 shRNA or control shRNA were subjected to Western blot analysis with anti-ALKBH4 and anti-β-actin antibodies. Uncropped Western blot data are shown in Supplementary Fig. S9. Representative pictures of three independent experiments are shown (upper pictures). Cell proliferation was detected using a xCELLigence DP system (lower panels). Degrees of cell proliferation expressed as cell index by the system are the means ± S.D. of three independent experiments (lower panels). (G) A549 cells stably expressing ALKBH4 shRNA (shALKBH4) and control shRNA (shControl), which are shown in (F), were subcutaneously injected into nude mice. The upper picture shows a xenograft tumour from mice inoculated with shControl- or shALKBH4-expressing A549 cells (shControl: n = 8; shALKBH4: n = 7). White scale bar, 1 cm. Tumour volume was calculated by measuring the tumour size every four days (lower panel). *p < 0.05 vs. control (Student’s t-test). (H) Tumour weight in shControl- and shALKBH4 xenografted mice. The values are presented as mean ± S.D. for each group. *p < 0.05 vs. shControl tumour (Student’s t-test).