Journal: Cell death & disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors.

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: Fig. 2 UBE3A promotes PBRM1 degradation in renal cancer cells. a 786-O cells were transfected with indicated plasmids for 24 h. Before harvested for western blotting analysis, cells were treated with or without 20 µM MG132 for 6 h. b 786-O cells were transfected with indicated plasmids. Twenty-four hours post transfection, cells were harvested for western blotting analysis. c 786-O cells were transfected with indicated plasmids. Twenty-four hours post transfection, cells were harvested for western blotting analysis. d 786-O cells were infected with indicated shRNAs. After 72 h, cells were treated with cycloheximide (CHX), and cells were collected for western blot analysis at different timepoints. e 786-O cells were transfected with indicated plasmids. After 24 h, cells were treated with CHX, and cells were collected for western blot analysis at different timepoints. f 786-O cells were infected with the indicated shRNAs. After 72 h, cells were collected for western blotting after treatment with MG132 for 8 h. g 786-O cells were transfected with the indicated plasmids. After 24 h, cells were collected for Western blotting after treatment with MG132 for 8 h. h 786-O cells were transfected with the indicated plasmids. After 24 h, cells were collected for western blotting after treatment with MG132 for 8 h.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Transfection, Western Blot, Infection

Journal: Cell death & disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors.

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: Fig. 7 A hypothesis model depicted that PKA phosphorylated UBE3A to prevent UBE3A degrading PBRM1. DAPK3 competed with PKA to bind with UBE3A and enhance the PBRM1 degradation in renal cancer cells. PBPJ transcriptionally regulated DAPK3 expression and then promoted UBE3A-mediated degradation of PBRM1. Then, PBRM1 increased the p21 expression and sensitized renal cancer cells to CDK4/6 inhibitors. In combination with RBPJ inhibitors, CDK4/6 inhibitors synergistically enhanced renal cancer cells.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Expressing

Journal: Oncotarget

Article Title: Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

doi: 10.18632/oncotarget.18712

Figure Lengend Snippet: (A) qRT-PCR analysis of pre-spliced hTERT expression levels (mean ± S.E n=3) within PB1-human chromosome 3 (PB1-#3fragment) hybrid clones, relative to wild-type PB1 cells. (B) FISH analysis of a single mouse A9-clone 8#3fragment hybrid generated by retro-transfer of the #3 fragment from clone 8 into mouse A9 cells. Representative DAPI-stained metaphase spreads of mouse A9-clone 8#3fragment clones, hybridised with, (i) TexasRed-labelled total human genomic paint and (ii) FITC-labelled chromosome 3-specific painting probes. (C) Summary of chromosome 3 microsatellite sequence-tagged site (STS) analysis of two mouse A9-clone 8#3 fragment hybrids (clones 4 and 9). A list of genes located within the 490kb region of deletion observed in clone 9 is indicated. (NRQ-Normalised relative quantity) Open circle represents loss of the marker while filled circles retained the marker.

Article Snippet: The plasmid vectors pCMV6AC- BAP1 (OriGene Technologies Inc., Rockville, MD, USA), pBABEpuro- BAF180/PBRM1 [ ], pCMVNeo-PARP-3 and associated empty vector controls were used to generate stable BAP1, PBRM1 and PARP-3, 21NT transfection clones, respectively.

Techniques: Quantitative RT-PCR, Expressing, Clone Assay, Generated, Staining, Sequencing, Marker

Journal: Oncotarget

Article Title: Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

doi: 10.18632/oncotarget.18712

Figure Lengend Snippet: (A) Graphical representation of the approximate positions of candidate genes within the 3p21.1-p21.3 region of human chromosome 3. Genomic coordinates of all candidate genes were obtained from the National Centre of Biotechnology Information (NCBI) database. (B) Candidate gene copy number (CN) variation analysis of HMEC strains and a panel of nine breast cancer cell lines. (C) Real-time qPCR mRNA expression analysis for SETD2, PBRM1, BAP1 and PARP-3 in breast cancer cell lines and normal breast cells (HMEC’s).

Article Snippet: The plasmid vectors pCMV6AC- BAP1 (OriGene Technologies Inc., Rockville, MD, USA), pBABEpuro- BAF180/PBRM1 [ ], pCMVNeo-PARP-3 and associated empty vector controls were used to generate stable BAP1, PBRM1 and PARP-3, 21NT transfection clones, respectively.

Techniques: Expressing

Journal: Oncotarget

Article Title: Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

doi: 10.18632/oncotarget.18712

Figure Lengend Snippet: qRT-PCR analysis of average (Ai) PARP-3 , (Bi) BAP1 , (Ci) SETD2 and (Di) PBRM1 expression levels (mean ± S.E n=3) and (Aii, Bii, Cii, and Dii) pre-spliced hTERT expression levels (mean ± S.E n=3) across five independent stable 21NT-candidate gene transfection clones and five independent stable 21NT-empty vector (EV) clones relative to parental 21NT cells (*p<0.05). Figure 3C shows (iii) SETD2 and (iv) hTERT expression levels (mean ± SD) within 21NT cells 48-144 hours following transient transfection of 21NT cells with pCMVNeo (EV) and pCMVNeo- SETD2 vector constructs, expressed relative to untreated 21NT cells. (NRQ-Normalised Relative Quantity).

Article Snippet: The plasmid vectors pCMV6AC- BAP1 (OriGene Technologies Inc., Rockville, MD, USA), pBABEpuro- BAF180/PBRM1 [ ], pCMVNeo-PARP-3 and associated empty vector controls were used to generate stable BAP1, PBRM1 and PARP-3, 21NT transfection clones, respectively.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Clone Assay, Plasmid Preparation, Construct

Journal: Oncotarget

Article Title: Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

doi: 10.18632/oncotarget.18712

Figure Lengend Snippet: Sequences of the synthetic oligonucleotides used as primers for qRT-PCR and thermal cycling parameters

Article Snippet: The plasmid vectors pCMV6AC- BAP1 (OriGene Technologies Inc., Rockville, MD, USA), pBABEpuro- BAF180/PBRM1 [ ], pCMVNeo-PARP-3 and associated empty vector controls were used to generate stable BAP1, PBRM1 and PARP-3, 21NT transfection clones, respectively.

Techniques: Sequencing

Journal: Microbiology Spectrum

Article Title: COG6 is an essential host factor for influenza A virus infection

doi: 10.1128/spectrum.01362-25

Figure Lengend Snippet: COG6 stabilizes IAV proteins. ( A ) WT and COG6-KO A549 cells were infected with PR8 virus at an MOI of 3, and viral RNA species of NP of PR8 virus were quantified by RT-qPCR at indicated time points. Viral RNA species were normalized to GAPDH mRNA levels. Data, presented as mean ± SD, are standardized to the corresponding RNA levels in WT cells from three independent biological replicates. ( B ) A minigenome assay was conducted in WT and COG6-KO H1299 cells co-transfected with vRNP reconstitution plasmids (pCAGGS-PB2, pCAGGS-PB1, pCAGGS-PA, and pCAGGS-NP), a reporter construct plasmid containing negative-sense Firefly luciferase gene flanked by the 5´ and 3´ ends of IAV NS1 gene, and a plasmid expressing Renilla luciferase (internal control). Firefly and Renilla luciferase activities were measured at 24 h post-transfection. The Firefly luciferase values are normalized to the Renilla luciferase values. Data are presented as mean ± SD from three independent experiments. ( C ) The cell lysates from ( B ) were analyzed for protein expression of PB2, PB1, PA, and NP using western blot. GAPDH served as a loading control. ( D–G ) Western blot analyses were performed on lysates from WT and COG6-KO H1299 cells, each individually transfected with vRNP reconstitution plasmids including pCAGGS-PB2 ( D ), pCAGGS-PB1 ( E ), pCAGGS-PA ( F ), pCAGGS-NP ( G ), or the vector control. GAPDH served as a loading control. ( H and I ) Western blot analyses were performed on lysates from WT and COG6-KO H1299 cells transfected with a plasmid encoding ZIKV NS1 protein ( H ) or the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein ( I ). GAPDH served as a loading control. Statistical analyses were determined using two-way analysis of variance with Sidak’s multiple comparisons test in ( A ) or unpaired, two-tailed Student’s t -test in ( B ). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: WT and COG6-KO H1299 cells were pre-seeded in 12-well plates and transfected with pCAGGS-NP, PB1, PB2, or PA for 24 h. Cells were treated with MG132 (25 μM, HY-13259, MedChemExpress), BafA1 (100 nM, HY-100558, MedChemExpress), or CQ (50 μM, HY-17589A, MedChemExpress).

Techniques: Infection, Virus, Quantitative RT-PCR, Transfection, Construct, Plasmid Preparation, Luciferase, Expressing, Control, Western Blot, Binding Assay, Two Tailed Test

Journal: Microbiology Spectrum

Article Title: COG6 is an essential host factor for influenza A virus infection

doi: 10.1128/spectrum.01362-25

Figure Lengend Snippet: COG6 prevents IAV proteins from lysosomal degradation. ( A–D ) Western blot analyses were performed on lysates from WT H1299 cells transfected with the vector control and COG6-KO H1299 cells transfected with vRNP reconstitution plasmids including pCAGGS-PB2 ( A ), pCAGGS-PB1 ( B ), pCAGGS-PA ( C ), or pCAGGS-NP ( D ). 24 h after plasmid transfection, the cells were treated with dimethyl sulfoxide, the proteasome inhibitor MG132, and the lysosomal inhibitors BafA1 or CQ. Additionally, COG6-KO H1299 cells were co-transfected with the pCAGGS-COG6 tagged with Flag (denoted as pCAGGS-COG6-Flag) and either pCAGGS-PB2, pCAGGS-PB1, pCAGGS-PA, or pCAGGS-NP. GAPDH served as a loading control. ( E ) WT and COG6-KO H1299 cells were mock- or infected with PR8 virus for 9 h at an MOI of 3. Cells were fixed and stained for LAMP1 in green, NP of PR8 virus in red, and nuclei in blue (DAPI). Scale bars, 50 µm. The relative fluorescence intensity of LAMP1 and the relative infection rates were analyzed by TissueFAXS. Over 1,000 cells were analyzed per biological replicate ( n = 3). Infection rates were normalized to WT. Statistical analyses were determined using two-way analysis of variance with Sidak’s multiple comparisons test. ****, P < 0.0001; ns, no significance.

Article Snippet: WT and COG6-KO H1299 cells were pre-seeded in 12-well plates and transfected with pCAGGS-NP, PB1, PB2, or PA for 24 h. Cells were treated with MG132 (25 μM, HY-13259, MedChemExpress), BafA1 (100 nM, HY-100558, MedChemExpress), or CQ (50 μM, HY-17589A, MedChemExpress).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Infection, Virus, Staining, Fluorescence

Journal: Microbiology Spectrum

Article Title: COG6 is an essential host factor for influenza A virus infection

doi: 10.1128/spectrum.01362-25

Figure Lengend Snippet: COG6 interacts with NP. ( A and D–F ) 293T cells were co-transfected for 24 h with pCAGGS-COG6-Flag along with either the vector control, pCAGGS-NP ( A ), pCAGGS-PB2 ( D ), pCAGGS-PB1 ( E ), or pCAGGS-PA ( F ). Cell lysates were immunoprecipitated using anti-FLAG M2 affinity gel and analyzed by immunoblotting with the indicated antibodies. ( B ) 293T cells were co-transfected for 24 h with pCAGGS-COG6 tagged with Myc (denoted as pCAGGS-COG6-Myc) and either pCAGGS-NP tagged with Flag and S (denoted as pCAGGS-NP-FS) or the vector control. Cell lysates were immunoprecipitated using anti-S tag affinity gel and analyzed by immunoblotting with the indicated antibodies. ( C ) The cell lysates co-transfected with the pCAGGS-COG6-Flag and pCAGGS-NP were treated with RNase A (100 µg/mL) at 4°C for 1 h prior to immunoprecipitation with anti-FLAG M2 affinity gel. GAPDH served as a loading control.

Article Snippet: WT and COG6-KO H1299 cells were pre-seeded in 12-well plates and transfected with pCAGGS-NP, PB1, PB2, or PA for 24 h. Cells were treated with MG132 (25 μM, HY-13259, MedChemExpress), BafA1 (100 nM, HY-100558, MedChemExpress), or CQ (50 μM, HY-17589A, MedChemExpress).

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot

Journal: Microbiology Spectrum

Article Title: COG6 is an essential host factor for influenza A virus infection

doi: 10.1128/spectrum.01362-25

Figure Lengend Snippet: The COG complex facilitates IAV infection by promoting viral attachment and protecting the viral proteins from lysosomal degradation. ( A ) Representative flow cytometric plots depicting frequencies of HA-positive cells from infected and uninfected cultures. WT, COG1-KO, COG2-KO, COG3-KO, COG4-KO, COG5-KO, COG6-KO, COG7-KO, and COG8-KO H1299 cells were infected with PR8 virus at an MOI of 3. At 12 h.p.i., cells were fixed, stained for IAV HA, and analyzed via flow cytometry. Data are presented as mean ± SD from three independent experiments. ( B ) Western blot analyses were performed on lysates from WT and COG6-KO H1299 cells transfected with vRNP reconstitution plasmids (pCAGGS-PB2, pCAGGS-PB1, pCAGGS-PA, and pCAGGS-NP) and COG6-KO H1299 cells following treatment with the lysosomal inhibitor BafA1. GAPDH served as a loading control. ( C ) Evaluation of the effects of WT, COG1-KO, COG2-KO, COG3-KO, COG4-KO, COG5-KO, COG6-KO, COG7-KO, and COG8-KO H1299 cells on PR8 attachment. PR8 virus at an MOI of 10 was incubated with cells on ice for 1 h. Viral RNA of attached PR8 virus was quantified by RT-qPCR and normalized to GAPDH mRNA levels. Data, presented as mean ± SD, are standardized to the corresponding RNA levels in WT cells from three independent biological replicates. Statistical analyses were determined using one-way analysis of variance with Dunnett’s multiple comparisons test in ( A and C ). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: WT and COG6-KO H1299 cells were pre-seeded in 12-well plates and transfected with pCAGGS-NP, PB1, PB2, or PA for 24 h. Cells were treated with MG132 (25 μM, HY-13259, MedChemExpress), BafA1 (100 nM, HY-100558, MedChemExpress), or CQ (50 μM, HY-17589A, MedChemExpress).

Techniques: Infection, Virus, Staining, Flow Cytometry, Western Blot, Transfection, Control, Incubation, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: New Insights into the Role of Polybromo-1 in Prostate Cancer

doi: 10.3390/ijms20122852

Figure Lengend Snippet: Comparison of the transcriptional levels of PBRM1 gene in PCa and BPH patients categorized according to their clinical and biochemical data. Odds ratio was calculated for each parameter among subjects classified as expressing low/high mRNA levels of PBRM1 based on median value of mRNA levels. The results for Gleason score were significant.

Article Snippet: Lentiviral particles encoding a scrambled shRNA sequence (1.0 × 10 5 IFU; shControl, sc-108080, Santa Cruz Biotechnology, Dallas, TX, USA) or a pool of lentiviral particles encoding four shRNAs specific for PBRM1 knockdown (1.0 × 10 5 IFU; shPBRM1, sc-76075-V, Santa Cruz Biotechnology) were used.

Techniques: Comparison, Expressing

Journal: International Journal of Molecular Sciences

Article Title: New Insights into the Role of Polybromo-1 in Prostate Cancer

doi: 10.3390/ijms20122852

Figure Lengend Snippet: Immunohistochemistry of PBRM1 in tissue microarrays (TMAs) containing prostate cancer tissues. The figure shows representative images of PCa patient-derived tissue microarrays (TMA). ( A ) PCa sample displaying a high Gleason score showing high intensity of total PBRM1 staining. ( B ) PCa sample displaying a low Gleason score sample showing low intensity of total PBRM1 staining. ( C ) PCa sample displaying a high Gleason score and showing a high nuclear and low cytoplasmic intensity of PBRM1 staining. ( D ) PCa sample displaying a low Gleason score showing low nuclear and high cytoplasmic/membranous intensity of PBRM1 staining. Arrows indicate the nuclear localization of PBRM1. ( E ) The table shows the correlation of PBRM1 with histopathological parameters of patients. * p < 0.05. PSA: Prostate-Specific Antigen. Scale bar = 20 μm and 20× magnification.

Article Snippet: Lentiviral particles encoding a scrambled shRNA sequence (1.0 × 10 5 IFU; shControl, sc-108080, Santa Cruz Biotechnology, Dallas, TX, USA) or a pool of lentiviral particles encoding four shRNAs specific for PBRM1 knockdown (1.0 × 10 5 IFU; shPBRM1, sc-76075-V, Santa Cruz Biotechnology) were used.

Techniques: Immunohistochemistry, Derivative Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: New Insights into the Role of Polybromo-1 in Prostate Cancer

doi: 10.3390/ijms20122852

Figure Lengend Snippet: Expression of mRNA levels of PBRM1 gene and localization of PBRM1 protein in the prostatic cell lines RWPE-1, LNCaP, DU-145, and PC-3. ( A ) mRNA relative levels of PBRM1 gene in RWPE-1, LNCaP, PC-3, and DU-145 cell lines. * p < 0.05; ** p < 0.01 ( B ) The cropped Western blotting of PBRM1 expression in membrane, cytoplasm, and nuclear fractions of RWPE-1, LNCaP, DU-145 e PC-3 cells. Full length PBRM1 (180 kDa) was detected. Actin and Lamin B were used as loading controls of cytoplasmic and nuclear extracts, respectively, and as controls of proper cell fractionation. ( C ) Immunofluorescent localization of PBRM1 was analyzed by staining cells with anti-PBRM1 (1:50, green), whereas nuclei were counter-stained with TO-PRO-3® (1:500, blue). Merged images (light blue) confirmed nuclear localization of PBRM1 in all cell lines. Cytoplasmic localization was only detected in prostate cancer cells. An intensive vesicular-like staining was observed in the cytoplasm of LNCaP, PC-3, and DU-145 cells. Arrows indicate nuclear localization of PBRM1. Three independent experiments were performed. Scale bar = 20 μm and 5 μm (magnification).

Article Snippet: Lentiviral particles encoding a scrambled shRNA sequence (1.0 × 10 5 IFU; shControl, sc-108080, Santa Cruz Biotechnology, Dallas, TX, USA) or a pool of lentiviral particles encoding four shRNAs specific for PBRM1 knockdown (1.0 × 10 5 IFU; shPBRM1, sc-76075-V, Santa Cruz Biotechnology) were used.

Techniques: Expressing, Western Blot, Membrane, Cell Fractionation, Staining

Journal: International Journal of Molecular Sciences

Article Title: New Insights into the Role of Polybromo-1 in Prostate Cancer

doi: 10.3390/ijms20122852

Figure Lengend Snippet: Analysis of PBRM1-downstream cellular events in PC-3 cells. ( A ) mRNA relative levels of PBRM1 gene in PC-3 cell line stably knocked down for PBRM1 (PC-3 shPBRM1) compared to a PC-3 cell line stably expressing a control shRNA (PC-3 shControl) ( B ) The cropped Western blotting of PBRM1 expression in membrane/cytoplasm (M + C) and nuclei (N) fractions of PC-3 shPBRM1 and PC-3 shControl cells. Full length PBRM1 (180 kDa) were detected. Actin and Lamin B were used as loading controls of cytoplasmic and nuclear extracts and as controls of proper cell fractionation. ( C ) EpCAM and ( D ) N-Cadherin were analyzed in PC-3 shPBRM1 and compared to PC-3 shControl cells by flow cytometry. ( E ) mRNA relative levels of TGF-β gene in PC-3 shPBRM1 and PC-3 shControl cells. ( F ) Migration potential was assessed by wound healing assay. PC-3 shControl and PC-3 shPBRM1 cells were plated, scratched with cell scraper, and photographed by phase-contrast microscopy. Representative images, showing cells migrated at 0 h and after 24 h. *** p < 0.001 is significant. Three independent experiments were performed. Scale bar = 400 nm.

Article Snippet: Lentiviral particles encoding a scrambled shRNA sequence (1.0 × 10 5 IFU; shControl, sc-108080, Santa Cruz Biotechnology, Dallas, TX, USA) or a pool of lentiviral particles encoding four shRNAs specific for PBRM1 knockdown (1.0 × 10 5 IFU; shPBRM1, sc-76075-V, Santa Cruz Biotechnology) were used.

Techniques: Stable Transfection, Expressing, Control, shRNA, Western Blot, Membrane, Cell Fractionation, Flow Cytometry, Migration, Wound Healing Assay, Microscopy