Fig. 2.

Validation of MLL1 as a negative mediator of trophoblast syncytialization. A mRNA levels of STB markers (HCG, Syncytin 1 and GCM1) in BeWo cells treated with MLL1 pathway inhibitors. The values are normalized to that of ACTIN and indicated as the mean ± SD (n = 3 biologically independent samples). B Western blots of Syncytin 1, GCM1, HCG, and H3K4me3 in BeWo cells exposed to 0, 5, and 10 μM MI-3454. C, D Immunostaining of E-cadherin (green) and DAPI (blue) (C) and corresponding quantification of multinucleated cells (D) among BeWo cells exposed to MI-3454 or DMSO. Scale bar, 40 μm. E–H mRNA levels of MLL1 and STB markers in BeWo cells treated with or without 25 μM FSK for 24, 48, and 72 h. I Western blots of MLL1, HCG, and H3K4me3 in BeWo cells treated with or without 25 μM FSK for 24, 48, and 72 h. J-N mRNA levels of MLL1 and STB markers (J-M) and western blots (N) of MLL1, Syncytin 1, GCM1, HCG, and H3K4me3 in BeWo cells transfected with si-MLL1 or si-NC. O-P Immunostaining of E-cadherin (green) and DAPI (blue) (O) and corresponding quantification of multinucleated cells (P) among BeWo cells transfected with si-MLL1 or si-NC. Scale bar, 40 μm. Data are presented as the means ± SD. **P < 0.01, *P < 0.05. CON, control; HCG, human chorionic gonadotropin; GCM1, glial cells missing transcription factor 1; H3k4me3, histone 3 lysine 4 trimethylation; H3 histone 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole; FSK, forskolin; si-NC, negative control small interfering RNA; si-MLL1, small interfering RNA targeting MLL1