Fig 2: The expression of ALKBH4 and E2F1of E2F1-target genes has a positive correlation in NSCLC clinical specimens. E2F1 (A) and E2F1-target gene ((B) CCNE1, (C) MYBL2, and (D) CDC25A) expression levels were measured using qPCR and were compared between normal and tumour tissues in NSCLC clinical specimens. Relative expression normalised to ACTB is shown. Data are represented as means ± S.D. ****p < 0.0001 for paired t-test. Correlation analysis was performed with the relative ALKBH4 expression and the relative E2F1 expression (E), CCNE1 expression (F), MYBL2 expression (G), and CDC25A expression (H) using 42-matched pairs of NSCLC samples. Pearson correlation analysis was conducted.

Fig 3: METTL3 and METTL14 promote the m6A modification of TRIM7 via the m6A reader YTHDF2. (a) The m6A levels in adjacent nontumorous (N) and osteosarcoma samples were measured. (b) Relative m6A levels of TRIM7 3′-UTR in HOS and MG63 cells were detected by RIP followed by qRT-PCR analysis. (c) TRIM7 3′-UTR enrichment was measured by RIP followed by qRT-PCR analysis in HOS and MG63 cells that were transfected with shRNAs targeting METTL3, METTL14, FTO, or ALKBH4. YTHDF2 silencing in HOS and MG63 cells increased TRIM7 mRNA levels (d) and stability (e). (f) RIP followed by qRT-PCR assay was performed to examine the interaction between YTHDF2 and 3′-UTR of TRIM7 mRNA. (g) A proposed molecular model of how increased TRIM7 expression, via inhibition of m6A modification, leads to the development and progression of osteosarcoma. All the experiments were repeated at least three times, and data are represented as mean ± SD. (a, e) *P < 0.05, ***P < 0.001 (two-way ANOVA followed by Bonferroni post-tests) compared with N or shNC. (c, d) ***P < 0.001 (one-way ANOVA followed by Bonferroni post-tests) compared with shNC. (f) ***P < 0.001 (unpaired Student's t-test) compared with IgG.

Fig 4: Association of ALKBH4 expression levels with recurrence-free survival and overall survival. (A) Expression of ALKBH4 in NSCLC specimens was examined using immunohistochemical staining. Representative results are shown. Black scale bars, 200 μm. Association of ALKBH4 expression levels with recurrence-free survival (B) and overall survival (C). NSCLC tissue specimens stained with anti-ALKBH4 were divided into two groups, according to ALKBH4 expression: negative (45 samples) and positive (35 samples). Data was statistically analysed using Log-rank test.

Fig 5: 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).