Fig 2: Interaction between FoxO1 and DNMT3A. (A) Representative mRNA levels of FoxO1 in the liver of 8-week-old mice (every group contains 10 mice); (B–D) FoxO1, DNMT3A gene expression in HepG2 cells treated with 0, 50, 100 nmol/L insulin concentration after 24 hours by RT-qPCR or Western Blot (n = 4 replicates/group in at least three independent isolations); (E) chip-qPCR analysis to detect binding between FoxO1 and DNMT3A. IgG was as negative control. (F, G) mRNA level of FoxO1 and DNMT3A after silence interfere FoxO1 on HepG2 cells (n = 4 replicates/group in at least three independent isolations); For RT-qPCR, Data were analyzed with the Eq. 2-ΔΔCT, where ΔΔCT = ΔCT (treatment group) - ΔCT (control group), and ΔCT = ΔCT (sample) - ΔCT (internal control). The values were normalized to ACTIN mRNA levels, ACTIN was an internal control. In all panels, data are presented as mean ± SD, *P < 0.05, **P < 0.01. Significance was determined by Student t test.

Fig 3: Methylation analysis of IGF2/H19 DMRs by pyrosequencing. (A) Schematic representation of mouse imprinted locus, showing the relative positions of the IGF2 and H19 genes and indicating the location of the four DMR knows to contribute to IGF2 imprinting. Enhancers (E) are indicated as black circles. The locations of the four DMRs within the IGF2/H19 imprinted locus represented by boxes are shaded to indicate preferential methylation of the maternal (M) or paternal (P) allel in each region. (B–E) Percentage of methylation for each CpG cytosine is calculated. (B) Methylation status of individual DNA strands of IGF2-DMR0 containing 8 CpG sites; (C) IGF2-DMRI containing 2 CpG sites; (D) IGF2-DMR2 containing 6 CpG sites; (E) H19-DMR containing 9 CpG and the average methylation ratio in each CpG site. (F) The relative gene expression of DNMTI, DNMT3A, DNMT3B in the liver of 8-week-old mice. Data were analyzed with the Eq. 2-ΔΔCT, where ΔΔCT = ΔCT (treatment group) -ΔCT (control group), and ΔCT = ΔCT (sample) -ΔCT (internal control). The values were normalized to ACTIN mRNA levels. For pyrosequencing and RT-qPCR, every group contains 10 mice. In all panels, data are presented as mean ± SD, *P < 0.05, **P < 0.01, Significance was determined by Student t test.

Fig 4: Methylation analysis of IG-DMR/MEG3-DMR by pyrosequencing. (A) Schematic representation of human imprinted locus, showing the relative position of the DLK1 and MEG3 genes and indicating the location of the two DMRs known to contribute to MEG3 imprinting. Exons are known as blue (DLK1 gene) and red (MEG3 gene) rectangles with arrows for transcription start sites. Under the genetic map listed specific gene sequence of the two DMRs. (B) Methylation status of individual DNA strands of IG-DMR containing 8 CpG sites and the average methylation ratio in each CpG site; (C) Methylation status of individual DNA strands of MEG3-DMR containing 8 CpG sites and the average methylation ratio in each CpG site. (D,E) The average methylation status of IG-DMR and MEG3-DMR, ten patients were included in each CpG site. (F,G) Dnmt3A, Dnmt3B RNA levels determined by RT-qPCR. (H) Dnmt3A, Dnmt3B protein levels determined by Western-Blot. Data were analyzed with the Equation 2−ΔΔCT, where ΔΔCT = ΔCT (treatment group) –ΔCT (control group), and ΔCT = ΔCT (sample)–ΔCT (internal control). The values were normalized to GAPDH mRNA levels. For western blot, the values were normalized to β-ACTIN protein levels. For pyrosequencing and RT-qPCR, there were 10 samples both for control and IVF group, for Western-blot, there were 3 samples both for control and IVF group. In all panels, data are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Significance was determined by Student t-test.

Fig 5: Prognostic significance of DNMT3A expression in 102 children with B-ALL. a ROC curve analysis of DNMT3A expression with relapse as an event. b The patients in DNMT3Alow group had poorer RFS than those in DNMT3A.high group (P < 0.001)