Fig 2: Bap1 ablation does not negatively affect stemness.(A) Western blot analysis of mouse trophoblast stem cells (mTSCs) grown in stem cell conditions (Stem) and upon 3 day differentiation in standard base medium (Base), or in base medium supplemented with FGF or conditioned medium (CM). (B) Western blot analysis assessing the dynamic changes in the stem cell markers CDX2 and ESRRB across a short-term differentiation time course in vector control compared to Bap1-mutant mTSCs (stem cell conditions = 0 h, and differentiation at 4, 8, and 24 hours (h)). Blots are representative of two independent replicates. (C) Proliferation assay of control and Bap1-/- mTSCs over 4 consecutive days. Bap1-/- mTSCs exhibit a significant increase in the proliferation rate compared to vector control cells (mean ± SEM; n = 3). p=0.0093; two-way ANOVA with Holm-Sidak’s multiple comparisons test. (D) RT-qPCR analysis of control and Bap1-/- mTSCs for stem cell and early differentiation marker genes. Stem cell markers are increased and the upregulation of differentiation markers delayed in Bap1-mutant mTSCs. Data are normalized to Sdha and displayed as mean of three biological replicates (i.e. independent clones) ± SEM; *p<0.05, **p<0.01 (two-way ANOVA with Sidak’s multiple comparisons test).

Fig 3: GFP-BAP1 proteomics show established BAP1 biology and validate HAT1 as a BAP1 interacting protein.(A and B). Volcanoplots showing GFP-BAP1 interaction partners for cytoplasmic (A) and nuclear fractions (B). (C). GFP-fusion constructs were expressed in HeLa cells and lysates were used in GFP immunoprecipitation. Immunoblot analysis using listed antibodies show HAT1 as a BAP1 interacting protein. (D) U2OS wt cell lysates were used for mock or BAP1 immunoprecipitation. Immunoblot analysis using listed antibodies confirms HAT1 interaction in U2OS cells.

Fig 4: Localization and expression testing of GFP-BAP1 variants in a ΔBAP1 background.(A). Confocal microscopy shows difference in localization and expression levels. Listed cell lines were grown on coverslips, stained using DAPI and Phalloidin-633 and mounted on microscopy slides. (B). Immunoblot analysis shows difference in expression levels as observed in (A). Cells were grown in presence of absence of doxycycline and lysates were analyzed using the listed antibodies. N-Stop: HeLa cells expressing only GFP (GFP-TEV-FLAG-3C-Stop linker).

Fig 5: Bap1 deficiency promotes epithelial-mesenchymal transition (EMT).(A) Colony morphology of wild-type (vector) and Bap1-mutant mouse trophoblast stem cells (mTSCs). Bap1-/- mTSCs show a fibroblast-like morphology with loss of cell-cell attachment compared to vector control mTSCs. Images are representative of five independent TSC clones each. Scale bar: 100 µm. (B) Principal component analysis of global transcriptomes of independent vector control (n = 3) and Bap1 knockout (KO) (n = 4) clones grown in stem cell conditions (0d) and after 3 days of differentiation (3d). (C) Gene ontology analyses of genes differentially expressed between vector and Bap1-mutant mTSCs in stem cell conditions. (D) Cell adhesion assay showing that Bap1-mutant mTSCs are less well attached to cell culture plastic compared to vector control cells. Data are mean of three independent replicates with three biological replicates ( = independent clones) per experiment. ***p<0.001 (Student’s t-test). (E) Morphology of 3D-trophospheres after 8 days of differentiation in low attachment conditions. Representative images of 2 independent vector control and Bap1 KO cell clones. Scale bar: 200 µm. (F) RT-qPCR analysis of EMT marker expression during a 6-day differentiation time course. Data are normalized to Sdha and displayed relative to vector in stem cell conditions (0d). Data are mean of five biological replicates (i.e. independent clones) ± SEM; *p<0.05, **p<0.01, ***p<0.001 (two-way ANOVA with Sidak’s multiple comparisons test). (G) Immunofluorescence analysis for CDH1 and F-actin (phalloidin) of vector control and Bap1-mutant mTSCs over 6 days of differentiation. Lack of BAP1 reduces cell-cell junctions (CDH1 staining) with a profound reorganization of the cytoskeleton (increased actin stress fibres). Data are representative of five independent vector control and Bap1 KO clones each. Scale bar: 100 µm. (H) Transwell invasion assay of vector control (V2, V4) and Bap1-mutant (clones A5, B10) mTSCs after 3 and 4 days of differentiation. Photographs of invasion filters show haematoxylin-stained cells that reached the bottom side of the filter after removal of the reconstituted basement membrane matrix (Matrigel). (I) Quantification of invaded cells, measured by colour intensity, normalized to 3-day controls. Data are mean of three independent replicates (three biological clones in each replicate) ± SEM; *p<0.05, **p<0.01** (two-way ANOVA with Sidak’s multiple comparisons test).