Fig 2: Luteal gene and protein expression of DNA methyltransferases (DNMT): DNMT1 (A,B); DNMT3A (C,D) and DNMT3B (E,F) in response to estradiol-17ß (E2) treatment in vivo and in gilts on day 12 of the estrous cycle and pregnancy. Control—gilts receiving placebo infusions into both uterine horns. E2—gilts receiving infusions of either placebo into one randomly selected horn or E2 (833 ng or 33.3 µg/infusion) into the contralateral horn. As referential groups, gilts on day 12 of the estrous cycle (D12 Cyclic) and pregnancy (D12 Pregnant) were included. Values of DNMTs gene expression were normalized against geometrical mean of ACTB and PPIA expression values (luteal samples from in vivo model) or PPIA expression values (luteal samples from day 12 pregnant/cyclic pigs). Values of DNMTs protein expression were normalized against total protein content. Main effect of treatment was detected on luteal expression of DNMT1 mRNA and protein, DNMT3A protein, and DNMT3B mRNA and protein (E2-treated groups vs. the control group; p < 0.05). There was no significant effect of the site of hormone administration (i.e., the placebo-treated uterine horn vs. the hormone-treated uterine horn within the same animal) on luteal expression of DNMT1, DNMT3A and DNMT3B mRNA and protein. Data are expressed as the mean ± SEM. Different letters indicate statistically significant differences between the control- and hormone-treated gilts (p < 0.05). Asterisk indicates statistical differences between gilts on day 12 of the estrous cycle and pregnancy (p < 0.05).

Fig 3: DNMT3B is required for endothelial cells to respond to Co-Cr. The expression levels and protein content of DNMT1 (a), DNMT3A (b), and DNMT3B (c) were investigated in the endothelial cells responding to Co-Cr_wo and Co-Cr_w, while the protein content for DNMT1 is depicted in (a’, representative image) and (a”, densitometry analysis), and DNMT3A is depicted in (b’, representative image) and (b”, densitometry analysis), and DNMT3B is depicted in (c’, representative image) and (c”, densitometry analysis). Statistics: * p < 0.05; *** p < 0.0002; **** p < 0.0001: Statistical differences when compared with the control group. * p < 0.05; ** p < 0.0004; *** p < 0.0003: Statistical differences when compared with Co-Cr_wo and Co-Cr_w. GAPDH was used as an internal control for sample loading (WB) and as a housekeeping gene (qPCR). The experiments were performed in triplicate (n = 3).