protein 1 bap1 status  (Santa Cruz Biotechnology)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transvitreal Retinochoroidal Biopsies of Primary Uveal Melanoma Reveal an Association of Low HLA Class I and High NK Cell Abundance in Low-Risk Disease

doi: 10.1167/iovs.66.2.24

Figure Lengend Snippet: HLA class I expression inversely associates with intratumoral NK cell percentages. ( A ) Bar plots showing the percentages of HLA class I–positive tumor cells ( top ) and the NK cells ( middle ) and CD8 T cells ( bottom ) of all immune cells per patient. ( B ) Box plot showing the percentage of HLA class I–positive tumor cells versus the BAP1 staining that was performed on tissue. ( C ) Box plot showing the percentage of HLA class I–positive tumor cells versus the risk group. ( D ) Kaplan–Meier plot showing the RFS of two groups of patients, separated by the median percentage of NK cells into NK high and NK low groups.

Article Snippet: To identify the BRCA-1 associated protein 1 (BAP1) status of tumors, a standard immunohistochemistry (IHC) staining using 1:100 anti-BAP1 (clone C-4, sc-28383; Santa Cruz Biotechnology, Dallas, TX, USA) was performed.

Techniques: Expressing, Staining

Journal: Investigative ophthalmology & visual science

Article Title: Transvitreal Retinochoroidal Biopsies of Primary Uveal Melanoma Reveal an Association of Low HLA Class I and High NK Cell Abundance in Low-Risk Disease.

doi: 10.1167/iovs.66.2.24

Figure Lengend Snippet: FIGURE 6. HLA class I expression inversely associates with intratumoral NK cell percentages. (A) Bar plots showing the percentages of HLA class I–positive tumor cells (top) and the NK cells (middle) and CD8 T cells (bottom) of all immune cells per patient. (B) Box plot showing the percentage of HLA class I–positive tumor cells versus the BAP1 staining that was performed on tissue. (C) Box plot showing the percentage of HLA class I–positive tumor cells versus the risk group. (D) Kaplan–Meier plot showing the RFS of two groups of patients, separated by the median percentage of NK cells into NKhigh and NKlow groups.

Article Snippet: To identify the BRCA1 associated protein 1 (BAP1) status of tumors, a standard immunohistochemistry (IHC) staining using 1:100 anti-BAP1 (clone C-4, sc-28383; Santa Cruz Biotechnology, Dallas, TX, USA) was performed.

Techniques: Expressing, Staining

Journal: Cancers

Article Title: Analysis of TERT mRNA Levels and Clinicopathological Features in Patients with Peritoneal Mesothelioma

doi: 10.3390/cancers17020252

Figure Lengend Snippet: Histological characteristics of mesothelioma patients’ biopsies relative to BAP1, p16 and MTAP.

Article Snippet: BRCA1-associated protein 1 (BAP1) (clone C4, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was classified as negative only if a complete absence of nuclear staining was observed in the presence of nuclear-positive lymphocytes.

Techniques:

Journal: Cancers

Article Title: Analysis of TERT mRNA Levels and Clinicopathological Features in Patients with Peritoneal Mesothelioma

doi: 10.3390/cancers17020252

Figure Lengend Snippet: Kaplan–Meier survival of patients with peritoneal mesothelioma. In ( A ), survival data is shown based on TERT score: patients with high TERT mRNA expression showed shorter survival times than their counterparts (violet lines); not statistically significant. In ( B ), survival data is shown based on histological type: epithelioid type (line blue), biphasic type (line red); differences are not significant. In ( C ), survival data based on mitotic index adjusted is shown: 2–4 (line red), 5–9 (line blue) or >10 (line green) mitosis expressed on 2 mm 2 ; p ≤ 0.0001. In ( D ), survival data based on sex is shown: male (line blue) and female (line red); p = 0.0152. In ( E ), survival data based on BAP1 is shown: positive (line blue) and negative (line orange); p = 0.0152. The dashed lines indicate SE.

Article Snippet: BRCA1-associated protein 1 (BAP1) (clone C4, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was classified as negative only if a complete absence of nuclear staining was observed in the presence of nuclear-positive lymphocytes.

Techniques: Expressing

Journal: Cancers

Article Title: Analysis of TERT mRNA Levels and Clinicopathological Features in Patients with Peritoneal Mesothelioma

doi: 10.3390/cancers17020252

Figure Lengend Snippet: Spearman’s correlation graphs between the customized score relative to the percentage of cells containing TERT mRNA (1+–4+ on the x -axis) and the score for a series of parameters obtained from morphological and cytogenetic analyses ( y -axis). In ( A ), TERT mRNA correlation with BAP1, p16 and MTAP protein expression, and with p16 cytogenetic alterations. In ( B ), TERT mRNA correlation with histotype, atypia and inflammatory infiltrate. In ( C ), TERT mRNA correlation with asbestous exposure type and time, sex, age and survival month. In ( D ), TERT mRNA correlation with necrosis, mitotic indexes, nuclear grade and ki-67. Statistical significance was determined by linear regression analysis. Only the significant r and p -values are reported.

Article Snippet: BRCA1-associated protein 1 (BAP1) (clone C4, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was classified as negative only if a complete absence of nuclear staining was observed in the presence of nuclear-positive lymphocytes.

Techniques: Expressing

Journal: Cancers

Article Title: Analysis of TERT mRNA Levels and Clinicopathological Features in Patients with Peritoneal Mesothelioma

doi: 10.3390/cancers17020252

Figure Lengend Snippet: Pathological factors predictive of peritoneal mesothelioma patient survival. Univariate analysis using log-rank test.

Article Snippet: BRCA1-associated protein 1 (BAP1) (clone C4, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was classified as negative only if a complete absence of nuclear staining was observed in the presence of nuclear-positive lymphocytes.

Techniques:

Journal: Acta Pharmaceutica Sinica. B

Article Title: Pharmacological inhibition of BAP1 recruits HERC2 to competitively dissociate BRCA1–BARD1, suppresses DNA repair and sensitizes CRC to radiotherapy

doi: 10.1016/j.apsb.2023.05.017

Figure Lengend Snippet: CRISPR-Cas9 screening identified BAP1 as a potential target of PT33. (A) Left: treatment scheme of DUBs sgRNA library screening experiment; right: relative abundance of individual genes from CRISPR-Cas9 screen. Genes with Log 2 FC > 1.5 and –Lg(FDR) > 2 were considered as potential target of PT33 to sensitize CRC cells to IR. (B) Colony formation assay detecting survival ratio of HCT116 cells knocking out corresponding genes treated with PT33 and IR. (C) PT33 binding to BAP1 was evaluated by cellular thermal shift assay. Upper and left below: HCT116 cells were treated with PT33 (5 μmol/L) for 1 h incubated in indicated temperature for 3 min; right below, HCT116 cells were treated with indicating concentrations of PT33 for 1 h and incubated in 52 centigrade for 3 min. Immunoblot detecting BAP1 intensity, GAPDH as loading control. (D) 3D presentation of the predicted binding mode of PT33 with BAP1 by molecular docking. Hydrophobic and hydrophilic residues of BAP1 were labeled by dashed lines (upper); and the surface is shown in below. PT33 is shown in green stick. (E, F) In vitro pull-down assays detecting PT33 and BAP1 covalent interaction. Purified myc-BAP1 was incubated with PT33 or PT33–Biotin. (E) BAP1 was immunoprecipitated and immunoblotted by biotin antibody. (F) Streptavidin pull down of biotin and immunoblotted by Myc antibody. (G) In vitro BAP1 deubiquitinating activity of BAP1 measured by UB-AMC hydrolysis assay. Upper: fluorescence-time curve; Below: the curve of covalent binding K obs to determinate the covalent inactivation rate ( k inact ) and reversible binding affinity K i for PT33 against BAP1. (H) HCT116 cells were treated with PT33 (125 nmol/L) for 12 h followed by 6 Gy IR, 6 h later, Flag-BAP1 was detected by IF assay. (H) HCT116 cells were treated as indicated and nuclear Flag-BAP1 was enriched and ultrafiltration, followed by Ub-AMC hydrolysis assay. Upper: representative fluorescence-time curve; below: statistical analysis of relative DUB activity. (B, C, G and I) Data are shown as mean ± SD ( n = 3). Statistical significance was determined by (B, I) Student's t test, (C) two-way ANOVA (n.s., not significant; ∗∗∗ P < 0.001).

Article Snippet: Figure 1 For the deubiquitinating activity assays of BAP1, recombinant BAP1 protein (No. ab268359, Abcam, Waltham, USA) or Flag-BAP1 purified from cell lysates using Anti-Flag Affinity Gel (No. P2271, Beyotime, Haimen, China) according to manufacturer's instructions was used.

Techniques: CRISPR, Library Screening, Colony Assay, Binding Assay, Thermal Shift Assay, Incubation, Western Blot, Control, Labeling, In Vitro, Purification, Immunoprecipitation, Activity Assay, Hydrolysis Assay, Fluorescence

Journal: Acta Pharmaceutica Sinica. B

Article Title: Pharmacological inhibition of BAP1 recruits HERC2 to competitively dissociate BRCA1–BARD1, suppresses DNA repair and sensitizes CRC to radiotherapy

doi: 10.1016/j.apsb.2023.05.017

Figure Lengend Snippet: Targeting to BAP1 with PT33 facilitates the K48-linked polyubiquitination of BRCA1. (A) BAP1 -WT or BAP1 -KO HCT116 cells were treated with PT33 (125 nmol/L) for 12 h and cycloheximide (CHX) for 2 h ahead of exposed to IR (6 Gy). BRCA1 levels at indicating time points after IR were detected by WB assay. Left: representative images; right: the relative BRCA1 levels, shown as the ratio of BRCA1 gray scale to Actin at indicated time normalized to that of 0 h. (B) BAP1 -WT and BAP1 -KO HCT116 cells were treated with PT33 for 12 h ahead of IR. IF staining for BRCA1 and γ -H2AX at the indicated time point. Left: representative images; right: quantification of colocalized foci. (C) HCT116 cells were treated with PT33 for 12 h and followed by IR. After 6 h, chromatin fractions were isolated and subject to immunoblotting. (D) BAP1 -WT and BAP1 -KO HCT116 cells were treated with PT33 for 12 h and followed by IR. After 6 h, the acid extracted fraction was isolated and subject to immunoblotting. (E) HCT116 cells expressing HA-Ubiquitin (WT, K6/K11/K27/K48/K63-specific) respectively were treated with PT33 for 12 h followed by IR (6 Gy); 6 h later, these cells were collected. WB assays for various HA-ubiquitin in cytoplasmic and nuclear fractions. (F) HCT116 cells expressing HA-Ubiquitin K6, K11 or K48 were treated with PT33 for 12 h followed by IR (6 Gy); 6 h later, these cells were collected. The nuclear lysate was subject to immunoprecipitation (IP) and WB assays. (G, H) IP and WB assays detecting the K48-ubiquitinated level of BRCA1. (G) Flag-BRCA1 and HA-Ubiquitin (K48) plasmids were transferred into BAP1 -WT or BAP1 -KO HCT116 cells following treatment with PT33 and then IR; (H) Myc- BAP1 (WT) or Myc- BAP1 (C91A) plasmid was transfected into HCT116 and treated with PT33 and then IR. DMSO or NonIR as control; PT33, 125 nmol/L; IR, 6 Gy. (A, B) The results are represented as cells in all views from 3 biological replicates. Quantitative data are shown as mean ± SD ( n = 3). Statistical significance was determined by two-way ANOVA (n.s., not significant; ∗∗∗ P < 0.001).

Article Snippet: Figure 1 For the deubiquitinating activity assays of BAP1, recombinant BAP1 protein (No. ab268359, Abcam, Waltham, USA) or Flag-BAP1 purified from cell lysates using Anti-Flag Affinity Gel (No. P2271, Beyotime, Haimen, China) according to manufacturer's instructions was used.

Techniques: Staining, Isolation, Western Blot, Expressing, Ubiquitin Proteomics, Immunoprecipitation, Plasmid Preparation, Transfection, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Pharmacological inhibition of BAP1 recruits HERC2 to competitively dissociate BRCA1–BARD1, suppresses DNA repair and sensitizes CRC to radiotherapy

doi: 10.1016/j.apsb.2023.05.017

Figure Lengend Snippet: BAP1 binding with PT33 recruits HERC2 to compete with BARD1 for BRCA1 interaction. (A) IP and WB assays were used to detect the interaction between Flag-BRCA1 and Myc-HERC2, BARD1 or BAP1. Flag- BRCA1 and Myc- HERC2 (3559–4834 aa) plasmids were transfected into BAP1 -WT or BAP1 -KO HCT116 cells following treatment with PT33 (125 nmol/L) for 12 h followed by IR (6 Gy), 6 h later, these cells were collected. (B) HCT116 cells with Flag- BRCA1 and Myc- HERC2 (3559–4834 aa) expression were exposed to IR; after 6 h, the cells were treated with PT33. At the indicated times, IP and WB assays were used to detect these interactions. (C) HCT116 cells were exposed to IR; after 6 h, the cells were treated with PT33. At the indicated times, IF assays for observing the distribution of BRCA1 and γ -H2AX. (D) BAP1 -WT and BAP1 -KO HCT116 cells were exposed to IR; after 6 h, the cells were treated with PT33. At the indicated times, IF assays for the distribution of BRCA1 and BARD1. (E, F) HERC2 was knocked down in BAP1 -WT and BAP1 -KO HCT116 cells with siRNA. (E) Cells were treated with PT33 and subject to HR reporter assay. (F) IF assay for distribution of RAD51. Cells were treated with PT33 (125 nmol/L) ahead of IR. At 6 h after IR, Nuclear foci were counted and the results are represented as cells in all views from 3 biological replicates. (E, F) Quantitative data are shown as mean ± SD ( n = 3). Statistical significance was determined by two-way ANOVA (n.s., not significant; ∗∗∗ P < 0.001).

Article Snippet: Figure 1 For the deubiquitinating activity assays of BAP1, recombinant BAP1 protein (No. ab268359, Abcam, Waltham, USA) or Flag-BAP1 purified from cell lysates using Anti-Flag Affinity Gel (No. P2271, Beyotime, Haimen, China) according to manufacturer's instructions was used.

Techniques: Binding Assay, Transfection, Expressing, Reporter Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Pharmacological inhibition of BAP1 recruits HERC2 to competitively dissociate BRCA1–BARD1, suppresses DNA repair and sensitizes CRC to radiotherapy

doi: 10.1016/j.apsb.2023.05.017

Figure Lengend Snippet: BAP1 deletion impairs PT33 sensitizing CRC to radiotherapy. (A, B) Representative images and statistical analysis of colony formation assays in BAP1 -WT and BAP1 -KO cells treated with PT33 (25 nmol/L) and IR at the indicated dose. Data are shown as mean ± SD ( n = 3; two-way ANOVA; n. s., not significant; ∗∗ P < 0.01 ∗∗∗ P < 0.001). (C, D) Representative images (upper) and quantitative analysis (below) of 3D-spheroid formation assays for BAP1 -WT and BAP1 -KO HCT116 (C) and HCT15 (D) cells treated as in Fig. 7 E. For spheroid number, data are mean ± SD ( n = 3); for spheroid diameter, data are all spheroids in each well from 3 independent biological replicates and shown as mean ± SD. Statistical significance was determined by two-way ANOVA (n.s, not significant; ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; blue symbols: compared to corresponding NonIR group; red symbols: compared to PT33 0 nmol/L + IR group). (E, F) BAP1 -WT and BAP1 -KO HCT116 xenografts were treated alternately with PT33 (1 mg/kg/day for 7 times, normal saline (NS) as control) and IR (1 Gy/day for 7 times). Representative images of tumors (E) and quantitative analysis of tumor volume (F). Data are mean ± SD ( n = 5/group). Statistical significance was determined by two-way ANOVA (n.s., not significant; ∗∗ P < 0.01).

Article Snippet: Figure 1 For the deubiquitinating activity assays of BAP1, recombinant BAP1 protein (No. ab268359, Abcam, Waltham, USA) or Flag-BAP1 purified from cell lysates using Anti-Flag Affinity Gel (No. P2271, Beyotime, Haimen, China) according to manufacturer's instructions was used.

Techniques: Saline, Control