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. 2023 Oct 23;51(21):11634–11651. doi: 10.1093/nar/gkad907

Figure 3.

Figure 3.

BRD9 regulates hESC maintenance and differentiation via controlling TGF-β and Wnt signaling pathway activity. (A) Global gene expression profiles of H1 cells upon I-BRD9 treatment for 12, 24 and 48 h. (B) GO analysis of down-regulated genes in hESCs treated with I-BRD9 for 12 h. (C) Transcript levels of TGF-β pathway-associated genes in hESCs treated with I-BRD9 for 24 h. (D) Western blot analysis of total SMAD2 and p-SMAD2 protein levels in hESCs cultured in E8 and E6 medium treated with I-BRD9 for 24 h. (E and F) Transcript levels of TGFB2, NODAL, LEFTY1, LEFTY2 and NANOG genes in hESCs cultured in E8 medium treated with I-BRD9 with and without addition of TN [5.2 ng/ml of TGF-β (T) and 100 ng/ml of NODAL (N)] proteins for 24 h. (G) Western blot analysis of SMAD2 and p-SMAD2 protein levels in hESCs cultured in E8 medium treated with 10 μM I-BRD9 with and without NODAL for 24 h; GAPDH served as a loading control. (H and I) Transcript levels of TBXT, MIXL1, TBX6, MESP1, HAND1 and GATA2 in early mesoderm differentiated from hESCs cultured in E8 medium with 20 ng/ml BMP4 upon treatment with 10 μM I-BRD9 (H) or 100 nM dBRD9-A (I) with and without TNA [5.2 ng/ml TGF-β (T), 100 ng/ml NODAL (N) and 100 ng/ml ACTIVIN-A (A)] for 2 days. (J) Transcript levels of endoderm markers SOX17, EOMES, SOX7, GATA4 and GATA6 in endoderm differentiated from hESCs upon BRD9 inhibition with and without TNA for 3 days. (K) Transcript levels of marker genes in neuroectoderm and neural crest differentiated from hESCs cultured in E6 medium upon BRD9 inhibition with and without TGF-β for 4 days. (L) Transcript levels of WNT3 and WNT3A in hESCs treated with 100 nM dBRD9-A for 2 days. (M) Western blot analysis of total β-CATENIN and active β-CATENIN protein levels in hESCs cultured in E8 medium treated with I-BRD9 for 24 h. (N) qPCR analysis of transcript levels of TBXT and MIXL1 in early mesoderm differentiated from hESCs upon BRD9 inhibition with and without 2 ng/ml WNT3A for 2 days. (O) Transcript levels of SOX17 and EOMES in endoderm differentiated from hESCs upon BRD9 inhibition with and without 2 ng/ml WNT3A. (P) qPCR analysis of transcript levels of WNT3 in I-BRD9-treated hESCs with and without TGF-β (5.2 ng/ml), NODAL (100 ng/ml) and ACTIVIN-A (100 ng/ml) for 24 h. (Q) qPCR analysis of transcript levels of TGF-β-associated genes in hESCs cultured in E8 medium upon BRD9 inhibition with and without 2 ng/ml WNT3A for 2 days.