Journal: Science advances

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

doi: 10.1126/sciadv.aax1738

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

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

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

Journal: Science advances

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

doi: 10.1126/sciadv.aax1738

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

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

Techniques: Expressing, Injection, Migration, Control

Journal: Science advances

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

doi: 10.1126/sciadv.aax1738

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

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

Techniques: RNA Sequencing, ChIP-sequencing, Expressing

Journal: Science advances

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

doi: 10.1126/sciadv.aax1738

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

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

Techniques: Injection, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: Clinical and pathological data of patients of esophageal squamous cell carcinoma in this study

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Mutagenesis

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: (a) BAP1 mutation in a patient with esophageal squamous cell carcinoma. In tumor tissue, TTT, encoding phenylalanine, is altered to ATT, encoding isoleucine (arrowhead). A faint peak observed in sequence data of this tumor likely represents contaminated residual non-cancerous DNA, as indicated by MLPA analysis (b). (b) MLPA data for all 17 exons of BAP1 gene on 3p21.1 are displayed on the x -axis. The table in the (b) also displays peaks of eight control probes, and RASSF1 MYND10 HEX1 ROBO1 FHIT MITF RBM5 and MLH1 in the BAP1 -neighboring region on chromosome 3p. Probe position and peak height in the (b) are described in SALSA MLPA probemix P417-B1 BAP1 (MRC Holland, Amsterdam, the Netherlands). Log 2 ratio of MLPA data for each probe is indicated on the y -axis. (c) Schematic of BAP1 with the F170I mutation. UCH, HCF1, ULD and NLS stand for ubiquitin C-terminal hydrolase domain, HCF1-binding domain, UCH37-like domain and nuclear localization signal, respectively. The F170I substitution is indicated by X. (d) Conservation of BAP1 in the region containing the F170I mutation. Alignment of amino acid sequences from codons 159–182 in human BAP1 and its counterparts in other species as identified by BLAST ( http://www.genome.jp/tools/blast/ ). Codon 170 is boxed.

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Mutagenesis, Sequencing, Control, Ubiquitin Proteomics, Binding Assay

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: (a) Deubiquitination of HCF1 by BAP1. Plasmids as described were transfected into HEK-293T cells. Cell extracts were immunoprecipitated with anti-HA antibody, followed by western blot analysis with either anti-Myc or with anti-HA antibody. The expression level of HCF1 or BAP1 (wild-type or F170I mutant) was confirmed by western blot analyses with anti-HA or anti-FLAG antibody. (b) Statistical analysis in triplicate, ubiquitination of HCF1. Statistical significance is indicated by the asterisk ( P < 0.05, Student's t -test). (c) Deubiquitination of BAP1. Plasmids were transfected into HEK-293T cells. Cell extracts were immunoprecipitated with anti-BAP1 antibody, followed by western blot analysis with either anti-Myc or anti-BAP1. (d) Statistical analysis in triplicate, ubiquitination of BAP1. Statistical significance is indicated by the double asterisk (P < 0.01, Student's t -test).

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Mutagenesis, Ubiquitin Proteomics

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: Subcellular localization of wild-type BAP1 or F170I mutant. Exogenous BAP1, wild-type (Wt) or F170I mutant (F170I), was visualized by anti-FLAG antibody treatment, followed by incubation with Alexa Fluor 488-conjugated antibody. A representative result is shown in (a). Exogenous BAP1 was more highly expressed in the nucleus (N > C), expressed equally between the nucleus and the cytoplasm (C = N), or expressed more highly in the cytoplasm (C > N). In each sample at least 200 cells were counted (b).

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Mutagenesis, Incubation

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: Heat map analyses of gene expression profiles. MT and WT stand for F170I mutant and wild-type BAP1, respectively. Expression profiles were examined in two independent transfections, 1 or 2 (a). A total of 5840 genes, selected by principal component analysis from the values of the first component of the vector, excluding genes without genome annotation, were used for heat map analysis by adjusting the average expression level of each gene to 0 (b).

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Gene Expression, Mutagenesis, Expressing, Transfection, Plasmid Preparation

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: Immunohistochemical staining of BAP1 in surgically resected esophageal cancer tissues. Representative features are shown, under low magnification (upper; bar 50 μm) and high magnification (lower; bar 20 μm). We classified (a) as BAP1 negative, (b) as BAP1-positive. In eight nuclear BAP1-negative cases, BAP1 was strongly expressed within the cytoplasm (c).

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Immunohistochemical staining, Staining

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: Result of quantitative RT-PCR for TCEAL7 expression in wild-type BAP1 or F170I mutant BAP1-transfected HEK-293T cells. RT-PCR was repeated in triplicate for duplicated transfections. Each asterisk indicates statistical significance ( P < 0.001). TF1, TF2 and NS stand for transfection 1, transfection 2 and no statistical significance ( P = 1.0 between TF1 and TF2 by F170I; P = 0.43 between TF1 and TF2 by Wt), respectively.

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Quantitative RT-PCR, Expressing, Mutagenesis, Transfection, Reverse Transcription Polymerase Chain Reaction

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, MTT Assay, Mutagenesis, Functional Assay, Binding Assay, Protein Binding, XTT Assay, Concentration Assay, Expressing, 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, 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, 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, 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

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, Immunohistochemistry, Recombinant, Plasmid Preparation, Luciferase, Sequencing, shRNA, Mutagenesis, Software, Staining, Transfection