Fig. 1: DNMT3B-catalyzed SMAD7 DNA methylation correlates with poor prognosis of LAD patients.

a, b Analysis of SMAD7 expression in 517 cases of LAD tissue and 59 cases of adjacent normal lung tissue (a), and 59 pairs of human LAD tissue and adjacent normal lung tissue (b) in the TCGA LUAD datasets. c Kaplan-Meier analysis (Log-rank test) of the 5-year overall survival of LAD patients in the TCGA LUAD datasets, who were divided into low or high SMAD7 expression subgroups. d, e Correlation between SMAD7 mRNA levels and the average beta-values (DNA methylation level) of the selected 6 methylation probes in 460 cases of LAD tissues in the TCGA LUAD datasets (d), and analysis of the average beta-values of the 6 probes against LAD tissues and adjacent normal lung tissues in the TCGA LUAD datasets (e). f, g Correlation between SMAD7 mRNA level and the average DNA methylation level of SMAD7 in 5 cases of normal lung tissues and 28 cases of our collected LAD cohort (f), and analysis of the average DNA methylation level of SMAD7 in adjacent normal lung tissues and LAD tissues in the cohort (g). h, i Kaplan-Meier analysis (Log-rank test) of the 5-year overall survival (left panel) and recurrence-free survival (right panel) of LAD patients in the TCGA LUAD datasets, who were divided into low or high SMAD7 methylation level subgroups. j, k Western blotting (WB) and qPCR analyses showed the effect of silencing DNMT3A and DNMT3B on the protein and mRNA level of SMAD7 in A549 and HCC827 cells with indicated treatments. l Bisulfite-sequencing PCR (BSP) validated the DNA methylation levels of region 1 of SMAD7 gene locus in A549 and HCC827 cells with indicated treatments. m, n WB and qPCR analysis showed the effect of overexpressing DNMT3A or DNMT3B on the protein and mRNA level of SMAD7 in A549 and HCC827 cells with indicated treatments. o BSP validated the DNA methylation level of region 1 of SMAD7 gene locus in A549 and HCC827 cells with indicated treatments. For aforementioned WB, qPCR and BSP assays, cells were treated with TGF-β1 at final concentration of 5 ng/mL for 48 h before the indicated assays were performed (j–o). Error bars represent mean ± SD. Two-tailed unpaired Student’s t-test (a, e and g), two-tailed paired Student’s t-test (b), and two-way ANOVA multiple comparison analysis (k, l, n, and o), respectively, were used for statistical analysis. **P < 0.01; *P < 0.05; ns, not significant, P > 0.05.