Fig. 4: USP7 knockout results in substantially increased DNA methylation in the absence of DNMT3A/3B. | Cell Discovery

Fig. 4: USP7 knockout results in substantially increased DNA methylation in the absence of DNMT3A/3B.

From: USP7 negatively controls global DNA methylation by attenuating ubiquitinated histone-dependent DNMT1 recruitment

Fig. 4

a WB analysis of control and DNMT3A/3B-DKO HeLa cells. b The levels of genomic DNA methylation (mC) in control and DNMT3A/3B-DKO HeLa cells determined by HPLC. **P ≤ 0.01. c The levels of genomic DNA methylation (mC) in control and DNMT3A/3B-DKO HeLa cells with progressively increased culture time determined by HPLC. *P ≤ 0.05; NS, not significant. d WB analysis showing the levels of key proteins in DNA methylation and p53 in control, DNMT3A/3B-DKO, and DNMT3A/DNMT3B/USP7-TKO HeLa cells. e Analysis of DNMT1 and UHRF1 protein stability in DNMT3A/3B-DKO and DNMT3A/DNMT3B/USP7-TKO HeLa cells. The cells were treated with cycloheximide (100 μg/mL) in culture medium for various times as indicated. The WB data were quantified by ImageJ and shown as relative levels to control (0 h) in the lower panel. f The levels of genomic DNA methylation (mC) in control, DNMT3A/3B-DKO and DNMT3A/DNMT3B/USP7-TKO HeLa cells determined by HPLC. ***P ≤ 0.001; **P ≤ 0.01. g The levels of genomic DNA methylation (mC) in control, DNMT3A/3B-DKO and DNMT3A/DNMT3B/USP7-TKO HeLa cells determined by LC-MS. ***P ≤ 0.001. h Co-IP analysis showing the interaction between USP7 and key proteins in DNA methylation. The indicated plasmids were co-transfected into HEK293T cells for 48 h. The cells were harvested, lysed, and enriched with FLAG-M2 beads. The precipitated complexes were subjected to WB analysis.

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