Fig 2: Overexpression of GATA3 does not eliminate the DHA-mediated downregulation of vWF expression. (a) Relative GATA3 mRNA expression in human umbilical vein endothelial cells (HUVECs) transfected with empty vector (EV) or GATA3 plasmids as measured by quantitative real-time PCR. n = 3; **, P < 0.01. (b) Representative immunoblots and densitometric analysis of GATA3 and GAPDH in HUVECs transfected with EV or GATA3 plasmids. n = 3; *, P < 0.05. (c) Relative vWF mRNA expression in HUVECs transfected with GATA3 plasmids. n = 4; *, P < 0.05. (d) Representative immunoblots and densitometric analysis of vWF and GAPDH in HUVECs transfected with GATA3 plasmids. n = 3; *, P < 0.05. (e) Relative vWF mRNA expression in HUVECs transfected with GATA3 plasmids and treated with 25 µM DHA for 24 h. n = 4; *, P < 0.05. (f) Representative immunoblots and densitometric analysis of vWF and GAPDH in HUVECs transfected with GATA3 plasmids and treated with 25 µM DHA for 24 h. n = 3; *, P < 0.05

Fig 3: Gata3 silencing promotes differentiation in 661W cells. (A) Gata 3 silencing decreases the expression of pax6 while increases the expression of Tuj1 in 661W cells, as evidenced by Western blot. (B) The relative levels of the Gata3, pax6, and Tuj1 proteins in 661W cells are presented in a histogram. (C) Significant up-regulation of Tuj1 (cytoplasm, green) were observed in the Gata3-silenced cells (cell nucleus, red). (D) Decreased pax6 staining (cell nucleus, green) was observed in the Gata3-silenced cells (cell nucleus, red). The nucleus is labeled with DAPI (blue). Scale bars represent 30 µm (* p < 0.05).

Fig 4: Gata3 silencing induces the differentiation of 661W cells. (A) Gata3 silencing by an siRNA decreased Nestin expression (red, in the cell cytoplasm) while upregulating Map2 expression in 661W cells (green, in the cell cytoplasm). (B) Gata3 silencing profoundly increased the expression of both Map2 (A/B) and Map2 (C/D). (C), The relative levels of the Map2 (A/B) and Map2 (C/D) proteins in 661W cells are presented in histograms. (D) Gata3 silencing significantly promoted retinal neurite outgrowth from 661W cells. Scale bars represent 10 µm. The asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01).

Fig 5: hPSCs transiently gain the ability to form epithelial cavitating structures during the naive-to-primed transition(A) A scheme of human peri-implantation development and correspondent hPSC states (PreEPI, preimplantation epiblast; PostEPI-E and PostEPI-L, early and late postimplantation epiblast, respectively; PS, primitive streak).(B) Experimental setup. Partially primed hPSCs were treated with either an inhibitor of ALK4/5/7, MAPK, or their combination.(C) qRT-PCR for markers after differentiation in indicated conditions; results of two independent experiments.(D) Stitched images of the cells in 24-well plates after differentiation in indicated conditions and staining with Phalloidin.(E) Bright-field image of 3D epithelial cavitating spheres obtained in AP condition. Note that the spheres remain attached to the surface of culture plates.(F) Immunofluorescence for GATA3 in combination with E-cadherin and CDX2 in combination with POU5F1, of partially primed hPSCs differentiated in AP condition (“AP”) and undifferentiated control (“undiff”).(G) Experimental setup. hPSCs on different days of the formative transition were differentiated in AP.(H) Stitched images of scanned 24-well plates showing hPSCs after different periods of the formative transition differentiated in AP (4',6-diamidino-2-phenylindole (DAPI) staining).(I) Bright-field images of hPSCs after different periods of the formative transition and conventional H9 hPSCs differentiated in AP.(J) qRT-PCR for markers during the time course of AP treatment of naive and partially primed hPSCs.(K) Immunofluorescence of naive and partially primed hPSCs during the time course of differentiation in AP.See also Figures S3 and S4.