Fig 2: PFOTE changes the genomic 5mC content and nuclear accumulation of DNMTs in lung cancer cells lines(A) Cells were treated with OTE (5 μg/mL) or PFOTE (5 μg/mL) for 120 h; then genomic 5mC content analysis was conducted. 5azaC-treated cells were used as positive controls. (B–C) The amounts of nuclear DNMT1, DNMT3A, and DNMT3B were determined using western blotting at the indicated PFOTE (5 μg/mL) treatment times. (D) Normalization and quantification of the intensities of protein bands were performed as described in Figure 1.

Fig 3: The roles of retinoic acid (RA) in epigenetic regulation in granulosa cells during follicular development.a The expression of Ccnd2, Dnmt1, Tet2, Lhcgr, Stk36, and Trnau1ap in cultured granulosa cells. Granulosa cells were collected from ovaries of 3-week-old mice after treatment with eCG for 6 h and were cultured in the absence of serum. FSH (100 ng/ml). T, testosterone (10 ng/ml). RA (1 μM). Levels of mRNA were normalized to that of L19. Values are represented as the mean ± SEM (n = 3 biological replicates). The value of the control (without any factors) was set as 1 and the data are presented as fold induction. *Significant differences were observed between the control group and the RA and/or FSH + T treatment group (p < 0.05). b, c The percentage of cells that expressed lower levels of DNMT1 was significantly increased by RA but decreased by the alcohol dehydrogenase (ADH) inhibitor (4MP). b The dot plot graph of double-positive granulosa cells with DNMT1 and PI during follicular development. c The percentage of cells that expressed lower levels of DNMT1 at S-phase. The lower threshold of DNMT1 levels at S-phase was set as 5% of cells with lower DNMT1 expression in the control group (C; without any factors) (red line). The cells that expressed lower levels of DNMT1 are shown in the red box, and cells with higher DNMT1 expression are shown in the black box at S-phase. Values are represented as the mean ± SEM (n = 4 biological replicates). *Significant differences were observed between the control group and the RA treatment group in the presence of FSH + T or the FSH + T treatment group in the presence of 1% FBS (p < 0.05). Significant differences were observed compared with those treated with no 4MP (p < 0.05). Significant differences in percentage values were transformed into normally distributed numbers by angle transformation and then analyzed by one-way ANOVA. Tukey–Kramer was used as post hoc test.

Fig 4: Knockdown of DDX3 enriches DNMT3A binding on promoters of tumor-suppressive miRNAs.(a) DDX3 knockdown promoted overexpression of DNMT3A. Cell lysates (50 μg) of shLuc, shDDX3 #2 and shDDX3 #3 cells were analyzed by western blotting with antibodies against DDX3, DNMT3A, DNMT3B, DNMT1 and β-actin. β-actin was used as internal control. (b) Suppression of DDX3 is associated with down-regulation of DNMT3A mRNA expression. DNMT3A, DNMT3B, DNMT1 and GAPDH mRNA expressions in shLuc, shDDX3 #2 and shDDX3 #3 cells were analyzed by qRT-PCR. GAPDH was used as internal control. The amount of each transcript in shDDX3 #2 and shDDX3 #3 cells was transformed into fold change relative to that of shLuc cells. (c) Knockdown of DDX3 led to reduction of DDX3 binding, enhancement of DNMT3A recruitment and elevation of gene-silencing histone mark H3K27me3 on promoter regions of MIR200B, MIR200C, MIR122 and MIR145. shLuc, shDDX3 #2 and shDDX3 #3 cells were subjected to ChIP assay. Binding of DDX3, DNMT3A, DNMT3B, DNMT1 and status of H3K27me3 were expressed as the relative fold change to rabbit IgG binding on the corresponding genomic region, which were normalized with input in individual cell line. All experiments were repeated at least three times, and the error bar indicated ± 1 s.d. of the mean. Statistical analyses were carried out using t test (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig 5: Kinetic changes in the expression levels of genes involved in epigenetic regulatory mechanisms in mouse granulosa cells during follicular development.a The expression levels of epigenetic-related genes in granulosa cells. Levels of mRNA were normalized to that of L19. Data represents the mean ± SEM (n = 3 biological replicates). Significant differences were observed between SF and eCG at 0 h (p < 0.05). *Significant differences were induced by eCG injection compared with before eCG injection (0 h) (p < 0.05). SF, secondary follicles. b, c Temporal changes of DNMT1 (b) and TET2 (c) in granulosa cells of secondary follicles (SF) with multilayered granulosa cells and antral follicles before or after eCG stimulation. β-Actin was used as a loading control. The results are representative of three independent experiments. Immunoblots shown are representative of three independent experiments. In each experiment, the different protein samples were used (n = 3 biological replicates). Quantitative expression of DNMT1 and TET2 relative to the expression of β-actin (control), as determined by western blotting. The SF value was set as 1 and the data are expressed as fold induction. The values are the mean ± SEM (n = 3 biological replicates). Significant differences were observed between SF and eCG at 0 h (p < 0.05). *Significant differences were induced by eCG injection compared with before eCG injection (0 h) (p < 0.05). Full-length blotting images of DNMT1, TET2, and β-actin are shown in Supplementary Fig. 8. d The localization of DNMT1, TET2, and PCNA in secondary follicles (SFs) and antral follicles (AFs) of 3-week-old immature mice or preovulatory follicles (PFs) of ovaries collected 48 h after eCG injection. DNMT1 and TET2 were visualized with Cy3, PCNA was visualized with FITC, and nuclei were stained with DAPI in the ovaries of 3-week-old mice treated with or without eCG. All of different length scale bars show 50 μm.