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: Chembiochem

Article Title: Characterization of BRCA1‐Associated Protein‐1 (BAP1) Aggregation Properties Induced by Cancer‐Associated Mutations

doi: 10.1002/cbic.202500372

Figure Lengend Snippet: Aggregation kinetics and model analysis by ThT binding assay. A) Aggregation kinetics of WT, N78S, C91W, F81V, and G128R BAP1‐UCH variants. The samples were tested in triplicate, with concentrations ranging from 30 μM to 1 μM, which are shown as dark to light colors. B) Half‐time plot of aggregation kinetics. The x ‐axis represents sample concentration, and the y ‐axis represents aggregation half‐time. Triplicates used in calculating slopes to explain the relationship between sample concentration and aggregation half‐time are marked with black borders. C) The aggregation mechanism is based on a secondary nucleation‐dominated model. k n , primary nucleation rate constant, k + , elongation rate constant, k 2 , secondary nucleation rate constant; n c , reaction order of primary nucleation; and n 2 , reaction order of secondary nucleation.

Article Snippet: HeLa‐Kyoto cells (Research Resource Identified, RRID: CVCL_1922; kindly provided by Professor Eric Nigg, University of Basel, Switzerland) grown on glass coverslips were transfected with FLAG‐HA‐tagged BAP1 variant plasmids ( https://www.addgene.org/22539/ ) for 48 h, fixed in buffer containing 20 mM PIPES (pH 6.8), 0.2% Triton X‐100, 10 mM EGTA, 1 mM MgCl 2 , and 4% formaldehyde (PTEMF solution), and then washed with phosphate‐buffered saline containing 0.05% Tween‐20 (PBST).

Techniques: Binding Assay, Concentration Assay

Journal: Chembiochem

Article Title: Characterization of BRCA1‐Associated Protein‐1 (BAP1) Aggregation Properties Induced by Cancer‐Associated Mutations

doi: 10.1002/cbic.202500372

Figure Lengend Snippet: Oligomerization kinetics of BAP1‐UCH variants. The hydrodynamic radii (R h ) of BAP1‐UCH variants as a function of incubation time monitored by DLS.

Article Snippet: HeLa‐Kyoto cells (Research Resource Identified, RRID: CVCL_1922; kindly provided by Professor Eric Nigg, University of Basel, Switzerland) grown on glass coverslips were transfected with FLAG‐HA‐tagged BAP1 variant plasmids ( https://www.addgene.org/22539/ ) for 48 h, fixed in buffer containing 20 mM PIPES (pH 6.8), 0.2% Triton X‐100, 10 mM EGTA, 1 mM MgCl 2 , and 4% formaldehyde (PTEMF solution), and then washed with phosphate‐buffered saline containing 0.05% Tween‐20 (PBST).

Techniques: Incubation

Journal: Chembiochem

Article Title: Characterization of BRCA1‐Associated Protein‐1 (BAP1) Aggregation Properties Induced by Cancer‐Associated Mutations

doi: 10.1002/cbic.202500372

Figure Lengend Snippet: Correlation plots of experimental folding stabilities of BAP1‐UCH variants with respect to the theoretical prediction of folding stability and aggregation kinetics parameters. The enthalpies of unfolding of the first transition, ΔH , and the second transition, ΔH 2 , of BAP1‐UCH variants, previously derived from DSC measurements, are shown on the horizontal axes. The theoretical free energy differences between the mutant and WT, ΔΔG mut‐WT , predicted by FoldX, are shown on the X ‐axes of A,B). The identities of the individual mutants are indicated next to the data point. Asterisks highlight the main mutants discussed in this study. The primary nucleation rate in logarithmic scales is shown in filled circles in C,D), with their scales indicated on the left Y ‐axes. The reaction order of the primary nucleation is shown in open squares in (C) and (D), with their scales shown on the right Y ‐axes. The secondary nucleation rate in logarithmic scales is shown in filled circles in E,F), with their scales indicated on the left Y ‐axes. The reaction order of the secondary nucleation is shown in open squares in (E) and (F), with their scales shown on the right Y ‐axes. The error bars represent the standard deviation (SD) of five replicates for each parameter as reported in Table .

Article Snippet: HeLa‐Kyoto cells (Research Resource Identified, RRID: CVCL_1922; kindly provided by Professor Eric Nigg, University of Basel, Switzerland) grown on glass coverslips were transfected with FLAG‐HA‐tagged BAP1 variant plasmids ( https://www.addgene.org/22539/ ) for 48 h, fixed in buffer containing 20 mM PIPES (pH 6.8), 0.2% Triton X‐100, 10 mM EGTA, 1 mM MgCl 2 , and 4% formaldehyde (PTEMF solution), and then washed with phosphate‐buffered saline containing 0.05% Tween‐20 (PBST).

Techniques: Derivative Assay, Mutagenesis, Standard Deviation

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: BAP1 regulates SRC transcriptionally. (A) reverse-phase protein array (RPPA) data analysis of SRC in KIRC-TCGA (clear cell renal cell carcinoma, ccRCC) stratified by BAP1 and/or PBRM1 mutation status. (B) RPPA data analysis in UVM-TCGA (uveal melanoma, UM) stratified by BAP1 mutation status. (C,D) Representative western blotting analysis of BAP1-deficient ccRCC UMRC-6 cell lines (C) and UM UPMM2 cell lines (D) reconstituted with an empty vector (EV), wild-type BAP1 , or p.C91S catalytically inactive BAP1 mutant. (E) Kaplan-Meier curves of overall survival in KIRC-TCGA depending on SRC expression levels, stratified by quartile. Intermediate SRC expression is the combination of the second and third quartiles. (F,G) qRT-PCR analysis of SRC mRNA expression in these reconstituted BAP1-deficient ccRCC (F) and UM (G) cell lines. (H) chromatin immunoprecipitation (ChIP) to assess BAP1 occupancy on the SRC promoter. Error bars represent the average ± SE ( n = 3–4). HR, hazard ratio; CI, confidence interval; n.s ., non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 for the indicated comparisons by t -tests.

Article Snippet: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

Techniques: Protein Array, Mutagenesis, Western Blot, Plasmid Preparation, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: BAP1 loss suppresses autophagy. The autophagic activity of UMRC-6 cells reconstituted with an empty vector (EV) or BAP1 (wild-type or p.C91S mutant) was analyzed using the following methods: (A) western blotting, (B,C) quantification of GFP-LC3 puncta ( n = 50), (D) western blotting of samples treated with Bafilomycin A1 (BafA1; 100 nM, 3 h), (E,F) quantification of autolysosomes vs . autophagosomes, (G) the number of WIPI2 dots per cell, (H) quantification of HiBiT-LC3 luminescence, and (I) the kinase activity of BECN1-bound VPS34. (J-L) Autophagy in UMRC-6 cells reconstituted with an empty vector (EV), wild-type BAP1 , or multiple BAP1 mutants observed in tumors were analyzed by microscopy of GFP-LC3 puncta formation (J,K) and western blotting (L) in cells treated with Bafilomycin A1 (100 nM, 3 h) or the DMSO control (vehicle). Error bars represent the average ± SE of three independent experiments. n.s ., non-significant; **, p < 0.01; ***, p < 0.001 for the indicated comparisons by t -tests. Non-overlapping letters (e.g., “a” vs . “b”) represent significant differences ( p < 0.05) using ANOVA and Student-Newman-Keuls test. Scale bar: 20 μm.

Article Snippet: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

Techniques: Activity Assay, Plasmid Preparation, Mutagenesis, Western Blot, Microscopy, Control

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: BAP1 loss leads to SRC-mediated suppression of autophagy. The effects of SRC inhibition on autophagy were assessed by treatment with dasatinib, and samples were analyzed by (A) western blotting, (B) HiBiT-LC3 luminescence, (C) GFP-LC3 puncta and (D) their quantification ( n = 50), (E) number of autophagosomes and autolysosomes ( n = 50), (F,G) number of GFP-FYVE puncta ( n = 50) treated with 1 µM dasatinib for 24 h or DMSO control, as well as by SRC silencing (H,I) or overexpression of a V5-tagged SRC construct (J-L). Bafilomycin A1 was added at 100 nM for 3 h and dasatinib at 1 µM for 24 h. Error bars represent the average ± SE of three independent experiments. n.s ., non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 for the indicated comparisons by t -tests. Non-overlapping letters represent significant differences ( p < 0.05) by ANOVA and Student-Newman-Keuls test. Scale bar: 20 μm.

Article Snippet: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

Techniques: Inhibition, Western Blot, Control, Over Expression, Construct

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: SRC binds to and phosphorylates BECN1 to regulate autophagy and proliferation. (A) BECN1 immunoprecipitation (IP) in UMRC-6 cells that overexpress SRC -V5 or an empty vector control (EV), containing plasmids for either constitutive SRC knockdown (sh SRC ) or a scrambled control (shSc). The arrow indicates the SRC -V5 band, and the bands below correspond to the heavy chain of IgG. (B) SRC -V5 was immunoprecipitated from UMRC-6 cells that overexpress SRC -V5 and were transfected with FLAG-BECN1 (or the corresponding empty vector control). (C) BECN1 (or IgG control) IP of UMRC-6 cells, followed by western blot analysis of phosphorylated tyrosine residues. (D) BECN1 IP of UMRC-6 cells overexpressing SRC -V5 (or empty vector control, EV) along with BAP1 (or EV) treated with 1 µM dasatinib for 24 h or DMSO control (vehicle). Red arrows indicate the bands for phosphorylated BECN1 (top) and VPS34 (bottom). (E,F) in vitro kinase assays of FLAG-BECN1 (or vector control) using in vitro transcribed/translated HA-SRC (E) or a kinase-dead version (F). The arrows in (E) indicate IgG heavy chains and the red asterisks (*) indicate the phosphorylated (top) and total (bottom) BECN1. (G-K) UMRC-6 cells stably expressing wild-type (WT) FLAG-BECN1, tyrosine phosphorylation mutants (non-phosphorylatable [BECN1 Y229/233/352F, BECN1-3F] or phospho-mimetic [BECN1 Y229/233/352E, BECN1-3E]) or an empty vector control (EV) were analyzed for autophagic flux by the number of GFP-LC3 puncta (G,H), the number of autolysosomes and autophagosomes (I), western blotting (J) or cell proliferation by Hoechst staining of the nuclei (K). Baf A1, 100 nM Bafilomycin A1 for 3 h; IP, immunoprecipitation; WCL, whole cell lysate. Error bars represent the average ± SE ( n = 50). n.s ., non-significant; *, p < 0.05; ***, p < 0.001 by t -tests. Non-overlapping letters represent significant differences ( p < 0.05) by ANOVA and Student-Newman-Keuls test. Scale bar: 20 μm.

Article Snippet: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

Techniques: Immunoprecipitation, Plasmid Preparation, Control, Knockdown, Transfection, Western Blot, In Vitro, Stable Transfection, Expressing, Phospho-proteomics, Staining

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: Dasatinib and SW076956 synergistically induce autophagy and reduce tumor growth in ovo and ex vivo in patient-derived tumor organoids. (A-D) Representative tumors of chorioallantoic membrane (CAM) assay (A) and quantification of tumor growth (B) of tumors treated with 10 µM Tat-BECN1 peptide (TB1) or 10 µM Tat-scrambled control (TS) (A,B) or with dasatinib (dasa, 1 µM), SW076956 (SW07, 40 µM) or a combination of both (C,D) for 7 days. (E-G) western blot analysis of several tumors treated with the TB1 or TS peptides (E), dasatinib, SW07 or a combination (F), and a representative patient-derived tumor organoid treated with the indicated compounds (G); BafA1: bafilomycin A1, 100 nM, 3 h; Das+SW07, combination of 1 µM dasatinib and 40 µM SW076956 for 24 h. Error bars represent the average ± SE. *, p < 0.05; ***, p < 0.001, t -test. (H-M) ombination effects of treatment with different concentrations of dasatinib and SW076956 on the viability of patient-derived tumor organoids (PDTOs) of ccRCC with BAP1 loss (H), ccRCC PDTOs with wild-type BAP1 (K), UM PDTOs with BAP1 loss (I,J) and UM PDTOs with wild-type BAP1 (L,M). The synergy/antagonism effects were determined using a Loewe synergy model with Combenefit software from two (I-M) or three (H) independent experiments.

Article Snippet: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

Techniques: In Ovo, Ex Vivo, Derivative Assay, Membrane, Chick Chorioallantoic Membrane Assay, Control, Western Blot, Software

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: Dasatinib and SW076956 synergistically induce autophagy and decrease cell viability in vitro . (A-D) the effects of dasatinib (at nM concentration) and SW076956 (at µM concentration) treatments on autophagy in UMRC-6 cells were assessed by western blotting alone (left panel) or in combination (right panel) (A), HiBiT-LC3 luminescence (B), and the number of GFP-LC3 puncta ( n = 50) (C,D). Cells were treated with 1 µM dasatinib and/or 40 µM SW076956 for 24 h. Baf A1, 100 nM Bafilomycin A1 for 3 h. Error bars represent the average ± SE of three independent experiments. n.s ., non-significant; *, p < 0.05; ***, p < 0.001 by t -tests. Non-overlapping letters indicate significant differences ( p < 0.05) by ANOVA and Student-newman-keuls test. (E,F) the combination effects of dasatinib and SW076956 treatments on cell viability of UMRC-6 (E) or TFK-1 (F) cells reconstituted with empty vector (EV), wild-type BAP1 , or a p.C91S BAP1 mutant were quantified 72 h after treatment of serial dilutions of single and combined compounds for three independent experiments. The synergy/antagonism effects of dasatinib and SW076956 were determined using a Loewe synergy model with Combenefit software from three independent experiments.

Article Snippet: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

Techniques: In Vitro, Concentration Assay, Western Blot, Plasmid Preparation, Mutagenesis, Software

Journal: Autophagy

Article Title: Autophagy suppression via SRC induction represents a therapeutic vulnerability for BAP1 -mutant cancers

doi: 10.1080/15548627.2025.2535265

Figure Lengend Snippet: Schematic representation of the proposed mechanism and platform for stratifying BAP1-loss patients who could benefit from treatment with SRC inhibitors and autophagy inducers. Lethal cancers with BAP1 mutations suppress autophagy through the binding and phosphorylation of BECN1 by the proto-oncogene SRC. Treatments with SRC inhibitors and autophagy inducers exhibited synergism in vitro , in ovo and ex vivo in patient-derived tumor organoids (PDTOs) with BAP1 loss, paving the way for the treatment of BAP1-deficient cancers with a combination of autophagy inducers and kinase inhibitors. The BAP1 immunohistochemistry images were reproduced from [10] with permission from Springer Nature.

Article Snippet: The plasmids to stably express wild-type BAP1 (pBABE-hygro-BAP1-HA, Addgene #154020), catalytically-inactive p.C91S mutant BAP1 (pBABE-hygro-BAP1-C91S-HA, Addgene #154021) or an empty vector control (pBABE-hygro, Addgene #1765) were previously described [ ].

Techniques: Binding Assay, Phospho-proteomics, In Vitro, In Ovo, Ex Vivo, Derivative Assay, Immunohistochemistry

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: ( A ) Area under the curve (AUC) values for 15 malignant mesothelioma (MM) cells treated for 6 days with 94 compounds. Each dot indicates the AUC value for an individual cell line treated. AUC <0.7 is indicated by the red dotted line — only those compounds with ≥2 cell lines below this value were analysed for statistically significant associations with gene mutations. The AUC values for rTRAIL are indicated by the red asterisk. ( B ) A Welch t-test was used to test for significant pharmacogenomics interactions between the 94 single agents in the screen and the presence of driver mutations in any of 5 MM cancer genes. Each volcano plot circle corresponds to a significant gene–drug interaction whose position on the x-axis indicates the corresponding effect size. Both half-axes are positive; the right side (green circles) indicates the effect sizes of sensitivity associations, whereas the left side (red circles) corresponds with the effect sizes of resistance associations. The position on the y-axis indicates the statistical significance of the identified interaction. The size of a given circle is proportional to the number of samples in which the selected functional event involved in the corresponding interaction occurs. Specific examples of associations are indicated where the effect size is large (rTRAIL and BAP1 mutations) or highly significant (cisplatin and CDKN2A mutations). ( C ) Cell viability between wild-type BAP1 (wt BAP1) (n = 10) and mutant BAP1 (mt BAP1) (n = 5) MM lines following 6 days of treatment with rTRAIL (t-test; *p=0.015). ( D ) Cell viability data for 17 MM lines treated for 6 days with a concentration range of rTRAIL (0.4–50 ng/ml). MM lines are coloured according to their sensitivity pattern (green = sensitive ( S ); orange = partially sensitive (PS); red = resistant ( R )). *Indicates cell lines harbouring BAP1 mutations. ( E ) Immunoblot of BAP1 protein expression in BAP1 -mutant versus BAP1 -wild-type MM cell lines. Sensitivity to rTRAIL treatment is indicated as font colour: green ( S ); orange (PS); red ( R ).

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Functional Assay, Mutagenesis, Concentration Assay, Western Blot, Expressing

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: 72 hour cell viability results for 9 MM cell lines (4 BAP1 -mutant - green and five wild-type - red) treated with (A) cisplatin (B) pemetrexed (C) FAS receptor agonistic antibody CH11, (D) TNF-α and 5 μM LCL161 or (E) DR5 agonist MEDI3039 assessed by MTT assay.

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Mutagenesis, MTT Assay

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: ( A ) BAP1 -wild-type H2818, MPP-89, H2373 and H2869 MM lines were transduced with BAP1 (shBAP1) or empty vector (EV) shRNA. Immunoblot confirmed BAP1 knockdown in the BAP1 shRNA-transduced cells. Parental and transduced cells were treated with rTRAIL (1000 ng/ml) and cell viability assessed after 72 hr by MTT assay (t-test; ****p<0.0001). ( B ) The BAP1 -wild-type breast cancer line MDAMB-231 and the renal cell carcinoma (RCC) lines Caki-1 and BB65 were transduced with BAP1 (shBAP1) or empty vector (EV) shRNA. Immunoblot confirmed BAP1 knockdown in the BAP1 shRNA transduced cells. Parental and transduced cells were treated with rTRAIL (1000 ng/ml) and cell viability assessed after 72 hr by MTT assay (t-test; ****p<0.0001). ( C ) The rTRAIL-sensitive H226 MM line, which harbours a homozygous deletion of BAP1 , was transduced with either a GFP control, wild-type BAP1 or a mutant BAP1 containing an inactive functional domain: C91A — inactivating mutation of deubiquitinase catalytic site; ΔHBM — deletion of HCF-1-binding motif; T493A — inactivating mutation of FOXK2-binding site; ΔASXL — deletion of ASXL1/2 protein-binding site; ΔCTD — deletion of C-terminal domain containing nuclear localisation signal. These transduced lines were treated with 50 ng/ml rTRAIL and cell death assessed with XTT assay (one-way ANOVA; **p<0.01). ( D ) The parental and transduced H226 MM lines were treated with a concentration range (1–100 pM) of the small molecule death receptor agonist MEDI3039 and cell viability assessed with XTT assay. ( E ) The BAP1 -wild-type MPP-89 MM line was transduced with ASXL1 (shASXL1), ASXL2 (shASXL2) or empty vector (EV) shRNA. qPCR confirmed a decrease in ASXL1 and ASXL2 mRNA expression in the ASXL1 shRNA and ASXL2 shRNA-transduced cells, respectively . Parental and transduced cells were treated with a concentration range (1–100 pM) of MEDI3039 and cell viability assessed with XTT assay. ( F ) Differential gene expression of apoptosis regulator genes in the catalytically inactive BAP1-mutant (C91A) relative to the wild-type BAP1-transduced (wt BAP1) H226 cells. ( G ) Immunoblot of apoptosis regulator proteins in the catalytically inactive BAP1-mutant (C91A), inactive ASXL1/2-binding site BAP1-mutant (ΔASXL) or wild-type BAP1-transduced (wt BAP1) H226 cells. ( H ) Flow cytometry analysis of death receptor 4 (DR4) and 5 (DR5) cell surface expression in H226 cells transduced with the catalytically inactive BAP1 -mutant (C91A) or wild-type BAP1 (wt BAP1) and of BAP1 -wild-type H2818 MM cells transduced with BAP1 (KD) or empty vector (EV) shRNA. The values represent the median fluorescence intensity (MFI).

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Transduction, Plasmid Preparation, shRNA, Western Blot, Knockdown, MTT Assay, Control, Mutagenesis, Functional Assay, Binding Assay, Protein Binding, XTT Assay, Concentration Assay, Expressing, Gene Expression, Flow Cytometry, Fluorescence

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: Three BAP1 -wild-type MM cell lines ( A ) MPP-89, ( B ) H2869 and ( C ) H2818 were transduced with empty vector (EV) or BAP1 shRNA (shBAP1). Immunoblot confirmed BAP1 knockdown. The parental, EV and shBAP1 cells were treated with rTRAIL for 24 hr and cell death measured by Annexin V/DAPI flow cytometry assay.

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Transduction, Plasmid Preparation, shRNA, Western Blot, Knockdown, Flow Cytometry

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: The MDAMB-231 breast cancer cell line was transduced with empty vector (EV) or BAP1 shRNA (shBAP1). Immunoblot confirmed BAP1 knockdown. Cells were treated with ( A ) rTRAIL and ( B ) MEDI3039 and cell viability measured with MTT assay at 72 hr. ( C ) Cells were treated with rTRAIL for 24 hr and cell death measured with Annexin V/DAPI flow cytometry assay.

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Transduction, Plasmid Preparation, shRNA, Western Blot, Knockdown, MTT Assay, Flow Cytometry

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: Clear cell renal carcinoma cell lines, Caki-1 and BB65, were transduced either with either empty vector (EV) or BAP1 shRNA (shBAP1). Immunoblot confirmed BAP1 knockdown. Cells were treated with rTRAIL ( A and C ) or MEDI3039 ( B and D ) for 72 hr and cell viability measured by MTT assay.

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Plasmid Preparation, shRNA, Western Blot, Knockdown, MTT Assay

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: Bladder (RT4) and breast (HCC1187) cancer cell lines harbouring nonsense mutations in BAP1 show increased sensitivity to rTRAIL compared with renal cell carcinoma or bladder cancer cell lines harbouring missense (769P and RCC10RGB) or wild-type BAP1 (BB65RCC and SW1710). Cell viability was measured after 6 days of treatment with 100 ng/ml rTRAIL.

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques:

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: The rTRAIL-sensitive H2804(A) and H28(B) mesothelioma cell lines, which harbour mutations in BAP1, were transduced with wild-type BAP1 (wt BAP1) or BAP1 with an inactive deubiquitinase catalytic domain (C91A) and treated with a dose range of rTRAIL. Cell death was assessed with Annexin V/DAPI apoptosis assay.

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Transduction, Apoptosis Assay

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: ( A ) Immunoblot analysis of H2AK119Ub levels in the parental, GFP-, wild-type BAP1 (wt BAP1)-, deubiquitinase mutant BAP1 (C91A)- and ASXL-binding mutant BAP1 (ΔASXL)-transduced H226 MM cell lines. ( B ) Immunofluorescence images of H2AK119Ub staining in the parental, deubiquitinase mutant-transduced (C91A), ASXL-binding mutant-transduced (ΔASXL) and wild-type BAP1 -transduced H226 cell lines. ( C ) Quantification of immunofluorescence staining in 2B (normalised to cell number; one-way ANOVA; ***p<0.001).

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Western Blot, Mutagenesis, Binding Assay, Immunofluorescence, Staining

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: The proteins in the pathway are highlighted in green if the expression in mt BAP1 is significantly less than wt BAP1 and red if the expression in mt BAP1 is significantly more than wt BAP1.

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Expressing

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: BAP1 immunoblot status, nuclear BAP1 staining and rTRAIL sensitivity (50 ng/ml) of the 25 human early passage MM cultures.

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Western Blot, Staining, Expressing

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: ( A ) Mean cell viability effect between human early passage MM cell lines (positive nuclear BAP1 staining; n = 13 and negative nuclear BAP1 staining; n = 12) as assessed by immunohistochemistry following 3 days of treatment with rTRAIL (50 ng/ml) (t-test, p=0.0067). ( B ) Immunohistochemical images of tumour explants derived from three MM patients treated with either vehicle or rTRAIL for 24 hr. Explants were stained with anti-BAP1 and anti-cleaved PARP (marker for apoptosis) antibodies. ( C ) The percentage of cleaved PARP-positive cells in tumour explants derived from three patients and treated with either vehicle or 0, 50, 100 and 200 ng/ml of rTRAIL for 24 hr was scored based on the percentage of cells with nuclear cleaved PARP-positive staining. ( D ) Weights of tumour xenografts derived from BAP1 -wild-type (wt BAP1) versus catalytically inactive BAP1-mutant (C91A mt BAP1) transduced MM cells following treatment with either vehicle or TRAIL (600 μg per mouse) at the time of sacrifice (day 42) (t-test). ( E ) Serial bioluminescence imaging of BAP1 -wild-type (wt BAP1) and catalytically inactive BAP1 -mutant (C91A) MM xenografts in mice treated with either vehicle or TRAIL. Mice were imaged on day 0 (after tumour inoculation), day 13 (before TRAIL administration) and day 41 (time of sacrifice). The intensity of luminescence is denoted by colour: red - high luciferase signal (high tumour burden) and blue - low luciferase signal (low tumour burden). ( F ) A time-course of bioluminescence scores in BAP1 -wild-type (wt BAP1) versus catalytically inactive BAP1 -mutant (C91A) MM tumour xenografts. Bioluminescence was measured on days 0, 13, 19, 26 and 41, 15 min after injecting the mice with 0.2 ml luciferin intraperitoneally. The number of photons emitted per second indicates the tumour burden (two way ANOVA).

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Staining, Immunohistochemistry, Immunohistochemical staining, Derivative Assay, Marker, Mutagenesis, Imaging, Luciferase

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet: ( A ) Schematic of in vivo experimental protocol. Mice were injected with H226 cells transduced with wild-type BAP1 and luciferase or catalytically inactive BAP1 -mutant (C91A) and luciferase on the right and left flanks, respectively. Mice were divided into two groups, each of which received 600 μg TRAIL or vehicle 6 days a week (day 14–40). Tumour size was assessed longitudinally with bioluminescence imaging on days 0, 13, 19, 26 and 41. ( B ) Size of tumours derived from BAP1 -wild-type (wt) versus catalytically inactive (C91A) BAP1 -mutant (mt) MM cells following treatment with either vehicle or TRAIL (600 μg per mouse) at time of sacrifice (day 42). A centimetre scale is included in the photograph for comparison.

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: In Vivo, Injection, Transduction, Luciferase, Mutagenesis, Imaging, Derivative Assay, Comparison

Journal: eLife

Article Title: Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells

doi: 10.7554/eLife.30224

Figure Lengend Snippet:

Article Snippet: BAP1 (NM_004656) Human cDNA Clone , pCMV6-AC BAP1 plasmid , Origene, Rockville, MD , Cat# SC117256 , .

Techniques: Flow Cytometry, Control, Immunohistochemistry, Recombinant, Plasmid Preparation, Luciferase, Sequencing, shRNA, Mutagenesis, Software, Staining, Transfection

Journal: Turkish Journal of Biochemistry

Article Title: Expression levels of BAP1, OGT, and YY1 genes in patients with eyelid tumors

doi: 10.1515/tjb-2021-0160

Figure Lengend Snippet: Figure 1: Comparison of the expression levels of BAP1, OGT and YY1 genes in the tumor group and the control group.

Article Snippet: To prevent non-specific binding, sections were incubated for 10 min in Ultra V Block (Thermo Scientific, USA) at RT, and then treated with anti-BAP1 antibody (rabbit polyclonal IgG, EPP10790, Elabscience, USA) (1:100 dilution), anti-OGT antibody (rabbit polyclonal IgG, EAP0752, Elabscience, USA) (1:100 dilution), and anti-YY1 antibody (rabbit polyclonal IgG, EPM12542, Elabscience, USA) (1:100 dilution) overnight at +4 °C.

Techniques: Comparison, Expressing, Control

Journal: Turkish Journal of Biochemistry

Article Title: Expression levels of BAP1, OGT, and YY1 genes in patients with eyelid tumors

doi: 10.1515/tjb-2021-0160

Figure Lengend Snippet: Figure 2: ROC curves of the levels of BAP1, OGT and YY1 in patients with eyelid tumors.

Article Snippet: To prevent non-specific binding, sections were incubated for 10 min in Ultra V Block (Thermo Scientific, USA) at RT, and then treated with anti-BAP1 antibody (rabbit polyclonal IgG, EPP10790, Elabscience, USA) (1:100 dilution), anti-OGT antibody (rabbit polyclonal IgG, EAP0752, Elabscience, USA) (1:100 dilution), and anti-YY1 antibody (rabbit polyclonal IgG, EPM12542, Elabscience, USA) (1:100 dilution) overnight at +4 °C.

Techniques:

Journal: Turkish Journal of Biochemistry

Article Title: Expression levels of BAP1, OGT, and YY1 genes in patients with eyelid tumors

doi: 10.1515/tjb-2021-0160

Figure Lengend Snippet: Figure 3: BAP1(A–B), OGT (D–E), and YY1 (G–H) immunolocalization in BCC. Negative control (C–F–I). White arrow: positive cells in tumor area and black arrow: positive cells in stromal cells. Chromogen; AEC, contrast dye; Hematoxylin. The intensity of the proteins was determined at ×10, ×20 and ×40 magnification with semi-quantitative scoring.

Article Snippet: To prevent non-specific binding, sections were incubated for 10 min in Ultra V Block (Thermo Scientific, USA) at RT, and then treated with anti-BAP1 antibody (rabbit polyclonal IgG, EPP10790, Elabscience, USA) (1:100 dilution), anti-OGT antibody (rabbit polyclonal IgG, EAP0752, Elabscience, USA) (1:100 dilution), and anti-YY1 antibody (rabbit polyclonal IgG, EPM12542, Elabscience, USA) (1:100 dilution) overnight at +4 °C.

Techniques: Negative Control

Journal: Turkish Journal of Biochemistry

Article Title: Expression levels of BAP1, OGT, and YY1 genes in patients with eyelid tumors

doi: 10.1515/tjb-2021-0160

Figure Lengend Snippet: Figure 4: BAP1, OGT and YY1 staining scores in BCC tumor and control areas.

Article Snippet: To prevent non-specific binding, sections were incubated for 10 min in Ultra V Block (Thermo Scientific, USA) at RT, and then treated with anti-BAP1 antibody (rabbit polyclonal IgG, EPP10790, Elabscience, USA) (1:100 dilution), anti-OGT antibody (rabbit polyclonal IgG, EAP0752, Elabscience, USA) (1:100 dilution), and anti-YY1 antibody (rabbit polyclonal IgG, EPM12542, Elabscience, USA) (1:100 dilution) overnight at +4 °C.

Techniques: Staining, Control