Fig 2: Effect of zebularine on the DNMTs expression and DNA methylation in HepG2 cells.(A) The protein level of DNMT1, DNMT3a, and DNMT3b after zebularine treatment for 72 h at different concentrations. After treatment, the cells were harvested and western blot analysis was performed to detect the protein level of DNMT1, DNMT3a, and DNMT3b. GAPDH was used as a loading control. Data are the means ± SEM of results from at least three independent experiments. *p<0.05, compared to 0 μM. (B) Scatter plot of the average beta values at 485,415 CpG sites for zebularine-treated (y-axis) and control (x-axis) HepG2 cells (n = 3 for each group). Dots for CpG sites whose delta-beta value is >0.1 or <−0.1 are shown in green (35 [0.0072%] hypermethylated and 162 [0.033%] hypomethylated CpG sites).

Fig 3: USP7 interacts with EZH2 and is required for EZH2 mediated protein stability. (A) Co-IP detection of the interactions between USP7 and EZH2, and between USP7 and EZH2 interaction partners. Immunoprecipitation with IgG was used as a negative control. (B–D) The mutual dependence of EZH2 and USP7 on each other’s interaction with proteins detected. (B) Detection of knockdown efficiency of a USP7 short-hairpin RNA (shUSP7). β-ACT was used as a loading control for IB assays. (C) Influence of USP7 knockdown on the interactions between EZH2 and other proteins. Proteins were immunoprecicpitated with an EZH2 antibody from cells without and with shUSP7. (D) Effect of EZH2 knockdown on the interactions between USP7 and other proteins. Proteins were immunoprecicpitated with a USP7 antibody from cells without and with shEZH2. In (C,D), immunoprecipitation with IgG was used as a negative control. (E,F) Interdependence of EZH2 and USP7 on their ability to maintain protein level. (E) USP7 knockdown counteracted the enhancement of protein level induced by EZH2 overexpression. (F) Knockdown of EZH2 compromised increment of protein level induced by USP7 overexpression. In (E,F), b-ACT expression was detected as a loading control in IB assays. (G–L) Effect of USP7 knockdown on the ubiquitination of proteins. Proteins were immunoprecipitated with antibodies against DNMT1 (G), HDAC1 (H), LSD1 (I), β-CAT (J), SMAD2 (K), and SMAD4 (L), respectively, from cells co-transfected with shCtrl or shUSP7 and HA-tagged ubiquitin plasmid, and treated with MG132. Precipitated proteins were detected for their ubiquitination levels using an HA antibody. (M) A working model depicting the molecular mechanism by which EZH2 regulates neuron-like differentiation in cancer cells.

Fig 4: Reduction of DNMT1 induces histone demethylation of Zeb2 or KLF4 promoter in PCa cells.(A) Experimental design of CHIP qPCR assays in 5-Aza– or siDNMT1-treated PCa cells. (B) A scheme shows the primer sites at the promoter region from transcription start site of the human Zeb2 or KLF4 genomic locus. Percent respective input of H3K9me3 and H3K27me3 levels at the Zeb2 genomic locus by CHIP qPCR assays (C) in vehicle- or 5-Aza (5 μM)–treated PCa cells (PC3, DU145) and (D) in siControl- or siDNMT1-treated PCa cells (PC3, DU145). Percent respective input of H3K9me3 and H3K27me3 levels at the KLF4 genomic locus by CHIP qPCR assays (E) in vehicle- or 5-Aza–treated PCa cells (PC3, DU145) and (F) in siControl- or siDNMT1-treated PCa cells (PC3, DU145). The data in C–F are representative of mean ± SD (n = 3). P values are calculated by Student's t test. (G) A summary diagram shows the activation of chromatin by histone demethylation of H3K9me3 and H3K27me3 following 5-Aza or siDNMT1 treatment, resulting in the increase of Zeb2 and KLF4 transcription.

Fig 5: The effect of RX-3117 mediated DNMT1 down-regulation on DNA methylation and expression of target proteins (ratio to β-actin) in A549 and SW1573 NSCLC cells. A: Time dependency of DNMT1 down-regulation. Western blot analysis of cell lysates after exposure to 1 µM RX-3117 after 1, 8, 16, and 24 h; β-actin was an internal loading control. B: Confocal microscopy pictures of A549 cells stained for the nucleus with DAPI (blue) and for total DNA methylation with 5mC Alexa Fluor-488 (green). The upper panel shows a control of untreated cells, the lower panel shows cells treated for 24 h with 10 µM RX-3117. Staining was predominantly nuclear. C: Western blot analysis of the TSG encoded proteins, MGMT and E-cadherin upon exposure to increasing concentrations of RX-3117 (0.1, 1 and 10 µM). As a positive control 10 µM of DAC was used and an internal control of β-actin was included. D: Western blot analysis of the TSG encoded protein p16 in SW1573 cells upon exposure to increasing concentrations of RX-3117 (0.1, 1 and 10 µM). The lower band under the p16 protein band, however, is a non-specific band. As a positive control 10 µM of DAC was used and an internal control of β-actin was included. This concentration of DAC was chosen based on its IC50 value in these cell lines. The blots are original, representative for one out of 2–3 separate experiments.