Journal: Science advances

Article Title: BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers.

doi: 10.1126/sciadv.aax1738

Figure Lengend Snippet: Fig. 1. Loss of Bap1 during Xenopus development produces a distinctive phenotype. (A) Representative embryos analyzed at late gastrula (stage 12) following injection into one blastomere at the two-cell stage of escalating doses (7.5, 10, and 20 ng) of a morpholino targeting the 5′UTR of bap1 mRNA (Bap1MO) or a bap1 base pair mismatch control morpholino (Bap1MO-Ctrl). Below each bright-field image is a corresponding fluorescence image demonstrating fluorescein isothiocyanate as a lineage tracer for the injected side (green color). Depletion of Bap1 produces gastrulation failure, as evidenced by incomplete blastopore closure (arrows). Arrowheads indicate injected side. Panels show dorsal view, anterior down. (B) Summary of results of experiments described in (A), showing that depletion of Bap1 produces gastrulation failure ranging from mild (yellow) to severe (red) in a dose-dependent manner. (C) Representative embryos treated as above with 7.5 ng of Bap1MO, which eventually completed gastrula- tion and developed axial foreshortening and bending (arrow) starting at early tail bud stages, compared to uninjected sibling embryos. Panels show lateral view, anterior left, except the lower right panel, which shows dorsal view, anterior down. (D) Representative embryos treated as above with 7.5 ng of Bap1MO, which eventually completed gastrulation and were evaluated at stage 37 or 45, showing microphthalmia or anophthalmia (black arrows) starting at late tail bud stages, and proliferation of morphologi- cally immature melanoblasts with altered migration pattern (red arrows) starting at late tail bud and early tadpole stages. Panels show lateral view (except panels labeled dorsal view), anterior left. Arrowheads indicate the injected side. (E) Transverse sections through the head of a representative early–tadpole stage embryo stained with hematoxylin and eosin, following injection into one blastomere (D1.2) at the 16-cell stage with 7.5 ng of Bap1MO, showing disruption of eye development on the side injected with Bap1MO (right side, arrowhead), compared to normal eye development on the uninjected control side (left side). Dotted line indicates midline. (F) Normal histologic appearance of the eye at early–tadpole stage embryo [same orientation as (E)], with ocular structures indicated. RPE, retinal pigment epithelium. (G to I) Represent ative eyes at late tail bud/early–tadpole stage embryos showing mild (G), moderate (H), and severe (I) ocular malformation associated with injection of 7.5 ng of Bap1MO into blastomere D1.2 (which gives rise to retina, lens, and other eye structures) at the 16-cell stage. In severe cases, such as the example in (I), retinal tissue does not form, and the eye remains filled with yolk platelets (pink). (J) Whole-mount in situ hybridization (WISH) of indicated eye markers in embryos injected with 7.5 ng of Bap1MO and a lineage tracer into D1.2 at the 16-cell stage and analyzed at the indicated stages, showing aberrant development of ocular tissues in the absence of Bap1. Markers include ventx2 (dorsal retina, unaffected), pax2 (ventral optic stalk), mitf and dct [retinal pigment epithelium and uveal melanocytes (UMCs)], rx1 (ciliary marginal zone and photoreceptor), otx2 and rbpms (retinal ganglion cell layer), and -crystallin (lens). Panels show lateral view, anterior side left. Uninj., uninjected. St., stage. Scale bars, 250 m.

Article Snippet: UMCs with knockout of BAP1 were created by clonally isolating UMCs stably transduced with lentiviral particles encoding spCAS9 (Addgene plasmid no. 50661) and guide RNA against BAP1 (Addgene plasmid no. 64114) directing the CRISPR-mediated deletion of the first exon of the BAP1 gene.

Techniques: Injection, Control, Fluorescence, Migration, Labeling, Staining, Disruption, In Situ Hybridization

Journal: Science advances

Article Title: BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers.

doi: 10.1126/sciadv.aax1738

Figure Lengend Snippet: Fig. 2. Bap1 loss deregulates expression of pluripotency and lineage commitment genes. (A to P) Representative embryos injected with 7.5 ng of the Bap1MO morpholino into one blastomere at the two-cell stage (arrowheads indicate injected side) and then fixed and analyzed for mRNA expression of the indicated developmental genes by WISH at the specified stages (gastrula, stage 12; midneurula, stages 14 to 17; early tail bud, stage 24). Bap1-depleted embryos fail to silence pluripotency factors such as vent1/2 (orthologs of mammalian Nanog) and oct25 (ortholog of mammalian Oct4) (A to C) and fail to activate lineage commitment factors such as fzd7 (dorsal mesoderm and ectoderm), vegT and bra (axial mesoderm), myoD (muscle), keratin1 (non-neural ectoderm), sox2 (neural ectoderm), rx1 (early eye field), zic1 and msx1 (neural fold/prospective neural crest), and foxD3 and sox10 (neural crest) (D to N). Bap1 loss results in a failure of neural crest cell migration in sox10-expressing cells compared to the uninjected control (O and P). Panels show the dorsal-caudal view (A, B, and D to F), the dorsal view (C and G to N), or the lateral view (O and P), anterior side left. Dotted lines indicate midline. Arrowheads indicate injected side. Scale bar, 250 m.

Article Snippet: UMCs with knockout of BAP1 were created by clonally isolating UMCs stably transduced with lentiviral particles encoding spCAS9 (Addgene plasmid no. 50661) and guide RNA against BAP1 (Addgene plasmid no. 64114) directing the CRISPR-mediated deletion of the first exon of the BAP1 gene.

Techniques: Expressing, Injection, Migration, Control

Journal: Science advances

Article Title: BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers.

doi: 10.1126/sciadv.aax1738

Figure Lengend Snippet: Fig. 3. Loss of Bap1 abrogates the assembly of H3K27ac at promoters of key genes regulating lineage commitment and differentiation. (A) Heat map demon- strating the top 1000 most differentially expressed genes by RNA-seq between embryos at one-cell stage with 15 ng of either Bap1MO or Bap1MO-Ctrl and collected at late gastrulation (stage 12), when morphologic effects of Bap1 loss are first evident. (B) Gene set enrichment analysis (GSEA) plots demonstrating the most highly significant pathways represented by the differentially expressed genes associated with Bap1 loss. FDR, false discovery rate. (C) Heat maps of ChIP-seq data demonstrating global genomic occupancy of the indicated histone marks across all annotated genes in embryos treated as above with either Bap1MO or Bap1MO-Ctrl. TSS, transcription start site. (D) Violin plots summarizing ChIP-seq data restricted to differentially expressed genes. (E) ChIP-seq and RNA-seq tracks of representative lineage commitment genes that fail to assemble H3K27ac at promoters and to activate mRNA expression in Bap1-deficient embryos.

Article Snippet: UMCs with knockout of BAP1 were created by clonally isolating UMCs stably transduced with lentiviral particles encoding spCAS9 (Addgene plasmid no. 50661) and guide RNA against BAP1 (Addgene plasmid no. 64114) directing the CRISPR-mediated deletion of the first exon of the BAP1 gene.

Techniques: RNA Sequencing, ChIP-sequencing, Expressing

Journal: Science advances

Article Title: BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers.

doi: 10.1126/sciadv.aax1738

Figure Lengend Snippet: Fig. 4. Hdac4 is a key mediator of the Bap1-deficient phenotype. (A) Representative embryos injected at the one-cell stage with 7.5 ng of Bap1MO with or without 16 ng of a morpholino directed against Hdac4 (Hdac4MO) and analyzed at midneurula stage (stage 15, dorsal view, anterior to left) and early tail bud stage (stage 26, lateral view, anterior to left). (B) Summary of results at stages 15 and 26, showing substantial rescue of the Bap1-deficient phenotype with Hdac4MO. (C) Representative embryos treated as above and analyzed by WISH, demonstrating that failed induction of the indicated developmental genes in Bap1-deficient embryos is rescued by Hdac4MO. Caudal view, dorsal up. (D) ChIP–quantitative polymerase chain reaction (qPCR) for indicated gene promoters following ChIP for H3K27ac, confirming that failure to assemble H3K27ac at promoters in Bap1-deficient embryos can be rescued by depletion of Hdac4. Scale bars, 250 m.

Article Snippet: UMCs with knockout of BAP1 were created by clonally isolating UMCs stably transduced with lentiviral particles encoding spCAS9 (Addgene plasmid no. 50661) and guide RNA against BAP1 (Addgene plasmid no. 64114) directing the CRISPR-mediated deletion of the first exon of the BAP1 gene.

Techniques: Injection, Real-time Polymerase Chain Reaction

Journal: Cancer cytopathology

Article Title: Analysis of early pleural fluid samples in patients with mesothelioma: A case series exploration of morphology, BAP1, and CDKN2A status with implications for the concept of mesothelioma in situ in cytology.

doi: 10.1002/cncy.22548

Figure Lengend Snippet: Figure 1. All initial pleural fluid specimens were comprised of predominantly dispersed mesothelial cells, (A) which in some cases, were present in moderate numbers with some size variation (arrow) and occasionally enlarged nucleoli, in keeping with an atypical mesothelial population (Papanicolaou stain). (B) The accompanying cell block revealed a similar population of cells, which were confirmed to be mesothelial in origin (calretinin immunohistochemistry [IHC]) and, in retrospect, (C) demonstrated loss of nuclear BRCA1-associated protein 1 (BAP1) staining (arrow) (BAP1 IHC). (D) Fluorescence in situ hybridization on this case (patient 1) identified no homozygous deletion of cyclin-dependent kinase inhibitor 2A (CDKN2A) (2 red CDKN2A signals were accompanied by at least 1 green chromosome 9 centromere signal per abnormal nucleus). (E) A diagnostic biopsy taken 2 years later revealed epithelioid mesothelioma (H&E stain) with (F) BAP1 loss (BAP1 IHC). (G) Fluorescence in situ hybridization analysis performed on this sample demonstrated homozygous deletion of CDKN2A (indicated by the loss of 2 CDKN2A red signals in abnormal nuclei with at least 1 retained chromosome 9 green signal, as indicated by the arrows).

Article Snippet: BAP1 IHC staining was performed using the anti- BAP1 C4 monoclonal antibody from Santa Cruz Biotechnology at a dilution of 1:50, with an UltraView Universal DAB Detection Kit on a Ventana BenchMark ULTRA platform.

Techniques: Papanicolaou Stain, Blocking Assay, Immunohistochemistry, Staining, Fluorescence, In Situ Hybridization, Diagnostic Assay

Journal: Cancer cytopathology

Article Title: Analysis of early pleural fluid samples in patients with mesothelioma: A case series exploration of morphology, BAP1, and CDKN2A status with implications for the concept of mesothelioma in situ in cytology.

doi: 10.1002/cncy.22548

Figure Lengend Snippet: Figure 2. Five of the initial samples carried a benign diagnosis, such as that of patient 8 shown here. (A) The smear contained dispersed mesothelial cells, including some enlarged forms (long arrows; the short arrow indicates mesothelial cells within normal size limits) among macrophages, neutrophils, and lymphocytes (DiffQuik preparation). (B) The cell block contained a similar population of cells with the retrospectively performed BRCA1-associated protein 1 (BAP1) demonstrating intact nuclear staining (arrow) (BAP1 immunohistochemistry [IHC]). (C,D) The diagnostic sample demonstrated large clusters of malignant mesothelial cells with irregular, hyperchromatic nuclei, prominent nucleoli and moderate volumes of dense cytoplasm (Papanicolaou stain). (E) Mesothelial origin was confirmed by cytokeratin 5/6 IHC, among other stains, and (F) BAP1 was noted to be lost on the retrospectively performed stain (BAP1 IHC).

Article Snippet: BAP1 IHC staining was performed using the anti- BAP1 C4 monoclonal antibody from Santa Cruz Biotechnology at a dilution of 1:50, with an UltraView Universal DAB Detection Kit on a Ventana BenchMark ULTRA platform.

Techniques: Biomarker Discovery, Blocking Assay, Staining, Immunohistochemistry, Diagnostic Assay, Papanicolaou Stain

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) Volcano plot of ATAC-seq peaks identified in BAP1 KOs (n=4) compared to MCF10A-Cas9 controls (n=2) cultured as mammospheres. Blue and red-colored dots represent peaks with loss and gain of ATAC-seq accessibility (log2FC < 0 and log2FC > 0), respectively. (B) Genomic compartment and (C) chromatin state distribution of differential ATAC-seq peaks (FDR < 0.01) identified in BAP1 KOs compared to MCF10A-Cas9 cells cultured as mammospheres. Promoters are defined as regions 500 base pairs (bp) upstream of transcription start sites (TSSs). Kb: kilobases. Pc: Polycomb. (D) Gene ontology enrichment of genes associated with differential ATAC loss peaks (FDR < 0.01, log2FC < 0) falling 2kb upstream from TSSs in BAP1 KOs compared to MCF10A-Cas9 cells cultured as mammospheres. Top 10 enriched ontologies are shown (p adjusted < 0.05). (E) Differential transcription factor binding in BAP1 KO clones compared to MCF10A-Cas9 (Ctrl) cells cultured as mammospheres. Blue: 224 underrepresented motifs (differential binding score < -0.1, p < 0.01); red: 64 overrepresented motifs (differential binding score > 0.1, p < 0.01); and gray: motifs with no representative alteration (-0.1 ≤ differential binding score ≤ 0.1) in ATAC-seq peaks from BAP1 KOs. (F) H2AK119ub1 levels in MCF10A-Cas9 control cells (Ctrl) and two BAP1 knockout (KO) clones (Cl1 and Cl2) cultured in attachment. H3 levels were assessed as an internal control. (G) Distribution of H2AK119ub1 ChIP-seq peaks (attached cells, FDR < 0.05) in regions of ATAC loss (mammosphere cultures, FDR < 0.01, log2FC < 0) identified in BAP1 KOs compared to MCF10A-Cas9 cells. Regions within 2kb upstream and downstream of TSSs and transcription end sites (TESs), respectively, are shown. ChIP-seq reads were normalized with RPGC (reads per genome coverage) per bin method. ChIP-seq peak distribution was analyzed for MCF10A wildtype (MCF10A-WT, n=2), MCF10A-Cas9 controls (MCF10A-Cas9, n=2), and both BAP1 KO clones (BAP1 KO, n=4).

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: Cell Culture, Binding Assay, Clone Assay, Control, Knock-Out, ChIP-sequencing

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) Volcano plot of differential expression analysis in BAP1 KOs compared to MCF10A-Cas9 control cells cultured as mammospheres (n=2 for MCF10A-Cas9 control cells and n=4 for BAP1 KOs). Blue and red-colored dots represent differentially expressed genes (DEGs, FDR < 0.01) with reduced or increased expression (log2FC < 0 and log2FC > 0), respectively. (B) Gene ontology enrichment of downregulated (left) and upregulated (right) DEGs (FDR < 0.01) in BAP1 KOs compared to MCF10A-Cas9 cells cultured as mammospheres. Top 10 enriched gene ontologies are shown (p adjusted < 0.05). (C) Gene set enrichment analysis (GSEA) plots of the transcriptome of BAP1 KOs compared to MCF10A-Cas9 control cells cultured as mammospheres, using the indicated gene sets. NES: normalized enrichment score. (D) Cell cycle analysis by propidium iodide staining in unsynchronized MCF10A-Cas9 and BAP1 KO mammospheres. Each bar graph shows the percentage distribution of cells in each cell cycle phase: G0-G1, S and G2-M. Significance analysis performed by Dunnett’s test (n=3, * P < 0.05, ** P < 0.01, ns: not significant).

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: Expressing, Control, Cell Culture, Cell Cycle Assay, Staining

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) CRISPR/Cas9 screening approach used for the identification of epigenetic regulator genes (ERGs) involved in the acquisition of mesenchymal breast cancer stem cell (BCSC) markers in non-tumorigenic breast cells. Adapted from Halaburkova et al. 2020 . gRNA: guide RNA. (B) Representation of enriched ERG gRNAs (false discovery rate [FDR] < 0.05) identified in the mesenchymal BCSC-like population of MCF10A cells infected with the ERG gRNA library compared to the bulk of cells on the day of sorting (n=2 MCF10A-Cas9 expressing clones). (C) Venn diagram of ERGs showing single nucleotide alterations in BC patients (TCGA-BRCA) of different molecular subtypes. Top 7 mutated ERGs identified in the TNBC subtype (proportion of SNAs (pSNA) > 0.019) are highlighted. (D) Kaplan-Meier analysis of disease-free survival in BC patients (TCGA-BRCA) divided in high and low BAP1 gene expression groups. * P < 0.05, ** P < 0.01.

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: CRISPR, Infection, Expressing, Clone Assay

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) BAP1 protein expression levels in MCF10A-Cas9 control cells (Ctrl), a heterozygous BAP1 deletion clone (HET), and two BAP1 knockout (KO) clones (Cl1 and Cl2). GAPDH levels were assessed as an internal control. (B) Representative images at 10x magnification of single, size-normalized MCF10A-Cas9 control and BAP1 KO mammospheres cultured for 72h. Scale bars: 100 µm. (C) Representative images at 20x magnification of single, size-normalized MCF10A-Cas9 control and BAP1 KO mammospheres stained with hematoxylin and eosin show mammosphere architecture differences and cellular changes between BAP1 KOs and controls. BAP1 KOs show disorganized architecture and a variable amount of intracytoplasmic vacuolization and cellular decohesion, all noted at the periphery of the mammospheres. Scale bars: 100 µm. See also Supplementary Figure S1. (D) Top: distribution of CD44 high and low populations in MCF10A-Cas9 controls and BAP1 KO cells cultured as mammospheres. Significance analysis performed by Fisher’s exact test on absolute cell counts (n=3, **** P < 0.0001). Bottom: proportion of BCSC- and epithelial-like populations based on expression of CD44, CD24 and EpCAM in MCF10A-Cas9 controls and BAP1 KO cells cultured as mammospheres. Significance analysis performed by Dunnett’s test (n=3, * P < 0.05, ** P < 0.01, ns: not significant). (E) Expression of EMT-associated genes in MCF10A-Cas9 and BAP1 KO cells cultured as mammospheres. Significance analysis performed by Dunnett’s test (n=3, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: not significant).

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: Expressing, Control, Knock-Out, Clone Assay, Cell Culture, Staining

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) Proportion of DEGs (FDR < 0.01) showing differential (FDR < 0.01) ATAC-seq loss (log 2 FC < 0), gain (log 2 FC > 0) or neutral signal (no loss or gain) within 2kb upstream of their TSSs in mammosphere-cultured BAP1 KOs compared to MCF10A-Cas9. Down: downregulated; up: upregulated. (B) Gene ontology enrichment of downregulated genes (FDR < 0.01, log 2 FC < 0) showing ATAC loss (FDR < 0.01, log 2 FC < 0) within 2kb upstream of their TSSs in mammosphere-cultured BAP1 KOs compared to MCF10A-Cas9. Top 10 enriched ontologies are shown (p adjusted < 0.05). (C) Diamond plot of glycosylation-associated DEGs (FDR < 0.01, GO: 0070085) showing changes in chromatin accessibility within 2kb upstream of their TSSs in mammosphere-cultured BAP1 KOs compared to MCF10A-Cas9 controls. Red: ATAC gain; blue: ATAC loss. y axis: RNA-seq gene expression (log2FC). (D) and (E) Genome browser snapshot of ATAC-seq and H2AK119ub1 peaks in mammosphere-cultured BAP1 KOs and MCF10A-Cas9, at the MGAT4A and ST6GALNAC1 genes. Horizontal bars represent differential ATAC-seq (FDR < 0.01) and/or ChIP-seq (FDR < 0.05) peaks. (F) Heatmap of glycosylation genes showing consistent alteration in chromatin accessibility (ATAC-seq, 2kb upstream of TSSs, left), RNA expression (RNA-seq, middle) and protein expression (proteomics, right) in BAP1 KO mammospheres compared to MCF10A-Cas9 controls. Colors and numbers on the heatmap represent the log2FC between BAP1 KOs and controls for each omics analysis (blue: ATAC loss or downregulation), and asterisks indicate that the change is significant (* FDR < 0.01). Where more than one ATAC-seq peak was annotated to a specific gene, only the closest peak to its TSS is shown in the heatmap.

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: Cell Culture, RNA Sequencing Assay, Expressing, ChIP-sequencing, RNA Expression

Journal: bioRxiv

Article Title: Disruption of the epigenetic regulator BAP1 drives chromatin remodeling leading to the emergence of cells with breast cancer stem cell properties and aberrant glycosylation

doi: 10.1101/2024.12.12.628129

Figure Lengend Snippet: (A) Relative abundance of chromatographic peaks identified by N -glycan profiling of mammosphere-cultured MCF10A-Cas9 and BAP1 KO cells. Significance analysis performed by Dunnett’s test (n=4, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: not significant). Top most abundant peaks across samples are shown. GP: glycan peak. (B) O -linked N -acetylglucosamine (O-GlcNAc) expression levels in MCF10A-Cas9 controls (Ctrl) and two BAP1 knockout (KO) clones (Cl1 and Cl2) cultured as mammospheres. GAPDH levels were assessed as an internal control. (C) Top: schematic representation of the constructs used for BAP1 re-expression, containing an HA tag and hygromycin (Hygro) resistance. Representations of the wildtype (WT) and mutant BAP1 (C91S mutation) constructs are shown. Bottom: BAP1 protein expression levels in MCF10A-Cas9 controls (Ctrl), and two BAP1 knockout (KO) clones (Cl1 and Cl2) infected with BAP1 wildtype (BAP) and BAP1 C91S mutant (MUT) rescue plasmids. GAPDH levels were assessed as an internal control. (D) Left: distribution of CD44 high and low populations in BAP1 KO Cl1 and BAP1 rescue cells (wildtype: WT; and mutant: MUT) cultured as mammospheres. Significance analysis performed by Fisher’s exact test on absolute cell counts (n=2, **** P < 0.0001). Right: proportion of BCSC-like populations based on expression of CD44, CD24 and EpCAM in BAP1 KO Cl1 and BAP1 rescue cells (wildtype: WT; and mutant: MUT) cultured as mammospheres. Significance analysis performed by Dunnett’s test (n=2, * P < 0.05, ns: not significant). (E) Expression of glycosyltransferase genes C1GALT1 , GALNT3 , GCNT1 , MGAT4A and ST6GALNAC1 in MCF10A-Cas9 (Ctrl), BAP1 KO cells and BAP1 rescues (wildtype, +; and mutant, mut) cultured as mammospheres. Significance analysis performed by Dunnett’s test compared to control (Ctrl) sample (n=3, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: not significant).

Article Snippet: Wildtype and catalytically inactive (C91S) BAP1 plasmids were obtained from Addgene (#154020 and #154021) and packaged into retroviral particles using Phoenix cells and CalPhos Mammalian Transfection Kit (BD Biosciences), as previously described .

Techniques: Cell Culture, Expressing, Knock-Out, Clone Assay, Control, Construct, Mutagenesis, Infection