Fig. 3: BAP1 overexpression impairs extravillous trophoblast formation and function.

A Phase contrast images of EVTs showing aggregated colony morphology with reduced elongation in BAP1-OE cells versus control EVTs. Scale bars, 150 µm. B Western blot analysis of ASXL1, BAP1, GFP-BAP1, HLA-G and TUBULIN (loading control) in BAP1-OE hTSC and EVTs compared to control cells. Quantification of HLA-G/TUBULIN ratio relative to control cells growing in stem cell conditions is plotted on the right (mean ± SEM, n = 4 independent experiments; ****p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test. C RT-qPCR analysis of EVT-specific (HLA-G, ITGA5, PLAC8, FSTL3, NOTUM, MMP2) markers in BAP1-OE hTSC and EVTs compared to control cells (mean ± SEM, n = 4 independent experiments; *p < 0.05, **p < 0.01, ****p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test. D On the left, heatmap of stage-specific marker expression (row-scaled Z-scores) in BAP1-OE EVTs versus control EVTs, showing retention of early-EVT markers (DAB2, LBP) and failure to activate late-EVT (MMP2, FLT1) transcriptional programs. Gene clusters defined by Kim et al., 2024. On the right, schematic model of differentiation blockade, demonstrating how BAP1 overexpression prevents progression from early to late EVT states. E Volcano plot of differentially expressed proteins (DEPs) in BAP1-overexpressing EVTs compared control EVTS (red: upregulated proteins; blue: downregulated proteins; adjusted p < 0.05, FC > 1.5). F Gene ontology enrichment analysis of DEPs, highlighting pathways associated with upregulated (red) and downregulated (blue) proteins. EVT extravillous trophoblast, hTSC human trophoblast stem cell, OE overexpression.