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. 2019 Aug 26;33(11):12541–12553. doi: 10.1096/fj.201901213R

Figure 6.

Figure 6

Sin3a KD increases genome-wide DNA methylation and nuclear Dnmt1 content. A) Immunocytochemical analysis of 5mc and 5hmc in morulae. The intensity of 5mc, but not 5hmc, was increased in KD embryos (n = 3; 5–10 embryos were analyzed/group each time. *P < 0.05. B) Bisulfite sequencing analysis of DMRs of Peg3, H19, and Snrpn in KD embryos at the morula stage. C) qPCR analysis of well-characterized imprinted genes, including Mest, Peg3, maternally expressed 3 (Meg3), Snrpn, and Igf2 in morulae (n = 3 pools of 5–10 embryos each group. *P < 0.05. D–F) Immunocytochemical detection of Dnmt3a (D) and Dnmt3b (E, F). Sin3a deficiency results in no change in Dnmt3a but an increase of Dnmt3b signal intensity (F). G) Dnmt1 is distributed mainly in the cytoplasm (arrowheads) of control embryos but more nuclear Dnmt1 (asterisks) detected in KD embryos. The experiment was conducted 3 times and 5–10 embryos were analyzed per group each time.*P < 0.05. Scale bar, 25 µm. H) Immunoblot analysis shows Dnmt1 protein abundance was increased in response to Sin3a KD. Two replicates were performed, and similar results obtained.