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: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: (a) Analysis of amino acid enrichment for ASXL1 (1-645). By Composition Profiler, using SwissProt 51 dataset as background. (b) Net charge per residue for ASXL1 (1-645). (c) Diagram of ASXL1 (1-590) showing regions for BAP1 and BRD4 binding. Each short red vertical line indicates an Arg. (d) Summary of (1-590) mutants that we designed and their effects on three indicated properties. (e) co-IP for FLAG in 293T cells transfected with FLAG-(1-590) (various mutants)-EGFP, and blotting for indicated proteins.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: Residue, Binding Assay, Co-Immunoprecipitation Assay, Transfection

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: (a) Immunostaining for ASXL1 and H2AK119ub in 293T cells transfected as indicated. Transfected cells are circled. (b, f) Percent of transfected cells that showed H2Aub reduction. From 3 biological repeats, each with 187-280 transfected cells (b), or 69-231 transfected cells (f). (c) In vitro H2A deubiquitination using purified BAP1, ASXL1 (1-590) (mEGFP- or mCherry-tagged, in green or red, respectively), and ubH2A-nucleosome, followed by SDS-PAGE and Coomassie staining. ubH2A- (lane 7) and unmodified (lane 8) nucleosomes were loaded to show histone identity. Bottom plot: Percent of ubH2A in total H2A (= ubH2A+H2A) was quantified by Image J, from 3 independent assays. (d) Diagram of Δ(391-426), Δ+eIF IDR , of 1-590. Δ+eIF IDR is the 1-590 construct in which the 391-426 region was replaced by eIF IDR . (e) Representative images of 293T cells expressing indicated constructs.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: Immunostaining, Transfection, In Vitro, Purification, SDS Page, Staining, Construct, Expressing

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: (a) 293T cells expressing indicated EGFP-tagged ASXL1 G646Wfs*12 , its 25RA mutant, or BAP1-mCherry. (b) 293T cells co-expressing indicated EGFP-tagged ASXL1 G646Wfs*12 or its 25RA mutant with BAP1-mCherry. (c) In vitro condensation of 1.67 µM of EGFP-ASXL1 (1-590) or mCherry-BAP1 individually and mixed.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: Expressing, Mutagenesis, In Vitro

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: All panels are based on RNA-seq analysis from . (a) Dot plots for fold changes of all genes in cells co-transduced with BAP1 and the indicated ASXL1 constructs. (b) Volcano plots of genes differentially expressed in cells co-transduced with BAP1 and the indicated ASXL1 constructs. DEGs (Probability > = 0.85) are in red (higher is 10RA than in 1-590) or blue (lower in 10RA than in 1-590). (c) Venn diagrams showing overlap between genes that, compared to co-transduction with BAP1 and 1-590, were downregulated by BAP1 and control vector (salmon), and genes that were downregulated by BAP1 and (1-590) 10RA (cyan) in each repeat (Probability > = 0.85). P values by Fisher’s exact test. (d-f) The most significantly enriched Biological Processes (d), tissue/cell-specific gene signatures (e), and transcriptional regulatory networks (f), for the 297 genes that were upregulated by ASXL1 (1-590) but less efficiently by 25RA in both repeats, as defined in . By Metascape. P values by two-sided hypergeometric test. (g) Expression levels (normalized read counts in RNA-seq) of indicated myeloid inflammatory response genes in in cells co-transduced with BAP1 and the indicated ASXL1 constructs.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: RNA Sequencing, Transduction, Construct, Control, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: All panels are based on RNA-seq analysis from , but focused on transposable elements (TEs). (a) Heatmap showing relative expression levels of 103 differentially expressed TEs in cells co-transduced with BAP1 and the indicated ASXL1 (1-590) constructs, including 62 and 41 TEs that were upregulated and downregulated by 1-590 than control, respectively. TEs in pink boxes were upregulated by co-transduction with BAP1 and 1-590 compared to BAP1 and control vector, but were upregulated less efficiently by BAP1 and (1-590) 10RA. (b) Percent of TE family types in total TEs and that were up- or down-regulated by co-transduction with BAP1 and 1-590 compared to BAP1 and control vector. Retrotransposons include ERVs/LTRs, LINEs, and SINEs. DNA-based transposons are also shown. (c) Volcano plots for total ERVs, ERVKs, and total LINE/SINEs that were differentially expressed in cells co-transduced with BAP1 and 1-590 over co-transduced with BAP1 and control vector. The red dots in each plot represent the labeled type of TEs, and the grey dots represent other types of TEs in each plot. (d) Expression of representative ERVs in cells co-transduced with BAP1 and the indicated ASXL1 (1-590) constructs, as shown in Integrated genome viewer.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: RNA Sequencing, Expressing, Transduction, Construct, Control, Plasmid Preparation, Labeling

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: (a, b) Assays on cells from the mice transplanted with BM transduced with using NRAS G12V and vector control, ASXL1 (1-590), or its 10RA mutant, as indicated. (a) Counts of various types of blood cells as indicated in the peripheral blood of the recipient mice at 9 months after transplant. Each dot is a mouse. WBC, white blood cells. NE, neutrophils. LY, lymphocytes. MO, monocytes. EO, eosinophils. BA, basophils. RBC, red blood cells. Hb, hemoglobin. Data are median (horizontal line), 25–75th percentiles (box) and 1.5 times the interquartile range recorded (whiskers), and the lack dashed line indicates average. (b) Representative flow cytometry analysis on c-Kit and CD11b of BM from moribund mice. (c, d) Non-competitive transplant assays to show BM repopulation. Following the experimental scheme in (c), chimerism (by percentages of CD45.1 + and CD45.2 + cells) in the peripheral blood 29 weeks after transplant was analyzed by flow cytometry (d). From 4, 3, 3 mice that received 1-590, or (1-590) 10RA or 25RA, respectively (all with BAP1). Representative flow analyses are shown on the right.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: Transduction, Plasmid Preparation, Control, Mutagenesis, Flow Cytometry

Journal: bioRxiv

Article Title: Unleashed condensation by recurrent mutations of an epigenetic regulator promotes cancer

doi: 10.64898/2026.02.18.706652

Figure Lengend Snippet: (a) Volcano plots of ERVs and LINE/SINEs differentially expressed in cells co-transduced with BAP1 and the indicated ASXL1 (1-1067) constructs. The red dots in each plot represent the labeled type of TEs, and the grey dots represent other types of TEs in each plot. (b) Heatmap for the relative expression levels for the 72 TEs that were expressed more highly by ASXL1 (1-1067) 36EQ than by 1-1067 in RNA-seq analyses from both before and after colony formation. (c) Venn diagrams showing the overlap between the TEs that were expressed more highly by transduction of ASXL1 (1-1067) 36EQ (salmon) than by 1-1067 and TEs that were expressed more highly by ASXL1 (1-1067) 34DN (cyan) than by 1-1067. P values by two-sided Fisher’s exact test. (d) Venn diagrams showing the overlap between the TEs (salmon) that were expressed more highly by transduction of ASXL1 (1-1067) 36EQ or 34DN than by 1-1067, from before colony RNA-seq, and the TEs (cyan) that were induced less efficiently by ASXL1 (1-590) 10RA than 1-590, from RNA-seq in . P values by two-sided Fisher’s exact test.

Article Snippet: Antibodies for the following proteins were used: BAP1 (Cell Signaling, #13187, RRID: AB_2798143), ASXL1 N-terminal (Abcam, ab228009, No RRID but used in literature , , and its staining pattern completely overlaps with GFP signal in our FmEGFP-ASXL1 Y591X cells), ASXL1 C-terminal (Abcam, ab50817, RRID:AB_867743), BRD4 (Abcam, ab128874, RRID:AB_11145462), Ser2 phosphorylated RNA-Pol II (Abcam, ab193468, RRID:AB_2905557), HP1α (Cell Signaling, #2616, RRID:AB_2070987), H2AK119ub (Cell Signaling, #8240, RRID:AB_10891618), GAPDH (Millipore # MAB374, RRID:AB_2107445, mouse, 1:5000 for immunoblotting), FLAG (Sigma # A2220, RRID: AB_10063035), HA (Cell signaling, #3724, RRID: AB_1549585, Rabbit, 1:1000 for immunoblotting).

Techniques: Transduction, Construct, Labeling, Expressing, RNA Sequencing

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 BAP1-deficient cell lines UMRC-6, TFK-1, and UPMM2 were reconstituted with 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), via retroviral infection as previously reported [ , ].

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 BAP1-deficient cell lines UMRC-6, TFK-1, and UPMM2 were reconstituted with 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), via retroviral infection as previously reported [ , ].

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 BAP1-deficient cell lines UMRC-6, TFK-1, and UPMM2 were reconstituted with 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), via retroviral infection as previously reported [ , ].

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 BAP1-deficient cell lines UMRC-6, TFK-1, and UPMM2 were reconstituted with 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), via retroviral infection as previously reported [ , ].

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 BAP1-deficient cell lines UMRC-6, TFK-1, and UPMM2 were reconstituted with 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), via retroviral infection as previously reported [ , ].

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 BAP1-deficient cell lines UMRC-6, TFK-1, and UPMM2 were reconstituted with 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), via retroviral infection as previously reported [ , ].

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 BAP1-deficient cell lines UMRC-6, TFK-1, and UPMM2 were reconstituted with 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), via retroviral infection as previously reported [ , ].

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

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: 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