Journal: Molecular Cell

Article Title: Efficient Pre-mRNA Cleavage Prevents Replication-Stress-Associated Genome Instability

doi: 10.1016/j.molcel.2018.11.036

Figure Lengend Snippet:

Article Snippet: GFP primary antibody (rabbit) , Torrey Pines Biolabs , Cat# TP401 071519; RRID: AB_10013661.

Techniques: Recombinant, Modification, Protease Inhibitor, Mutagenesis, Transfection, Labeling, Purification, Multiplex Assay, Sequencing, Derivative Assay, Clone Assay, esiRNA, Negative Control, Software

Journal: Oncology Letters

Article Title: Role of high ubiquitin-conjugating enzyme E2 expression as a prognostic factor in nasopharyngeal carcinoma

doi: 10.3892/ol.2022.13314

Figure Lengend Snippet: Increased expression levels of UBE2B in NPC tissues. Analysis of the public datasets (A) GSE12452 and (B) GSE68799 indicated increased expression levels of UBE2B in NPC compared to normal nasopharynx tissues. The heatmaps (left panel) present UBE2B transcription levels in each tissue sample and the scatter plots (right panel) indicate gene expression compared between normal nasopharyngeal mucosa and NPC tissues. The GSE12452 dataset contained mRNA signals from 10 non-NPC and 31 NPC tissues and the GSE68799 dataset contained 4 non-NPC and 42 NPC tissues. NPC, nasopharyngeal carcinoma; UBE2B, ubiquitin-conjugating enzyme E2 B.

Article Snippet: The slides were then washed with tris-buffered saline and incubated with a primary rabbit polyclonal antibody against UBE2B (1:100 dilution; GeneTex, Inc.) for 1 h. A ChemMate EnVision kit (DAKO; Agilent Technologies, Inc.) was applied to detect the primary antibody.

Techniques: Expressing, Gene Expression, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: Role of high ubiquitin-conjugating enzyme E2 expression as a prognostic factor in nasopharyngeal carcinoma

doi: 10.3892/ol.2022.13314

Figure Lengend Snippet: UBE2B has an important role in the carcinogenesis of NPC cells. (A) Western blot analysis demonstrated that UBE2B expression levels were higher in TW01 cells than those in DOK cells. (B) Bar graphs indicated a higher UBE2B/actin ratio in TW01 cells compared with that in DOK cells. (C) Expression levels of UBE2B in NPC cells treated with control or UBE2B-targeting siRNA were determined using western blot analysis. (D) By using methylene blue staining, the numbers of colonies formed, which consist of at least 50 tumor cells, were manually recorded and compared. (E) Bar graphs indicated decreased numbers of formed colonies in UBE2B-deficient NPC cells as compared with UBE2B-proficient cells. At least three independent experiments were performed and values were expressed as the mean ± standard deviation. ****P<0.01 for NPC cells vs. DOK cells and siUBE2B vs. control. Fold changes in protein levels listed under each blot were normalized to the levels of the control counterparts and analyzed by using ImageJ densitometry analysis. NPC, nasopharyngeal carcinoma; UBE2B, ubiquitin-conjugating enzyme E2 B; siRNA, small interfering RNA.

Article Snippet: The slides were then washed with tris-buffered saline and incubated with a primary rabbit polyclonal antibody against UBE2B (1:100 dilution; GeneTex, Inc.) for 1 h. A ChemMate EnVision kit (DAKO; Agilent Technologies, Inc.) was applied to detect the primary antibody.

Techniques: Western Blot, Expressing, Control, Staining, Standard Deviation, Ubiquitin Proteomics, Small Interfering RNA

Journal: Oncology Letters

Article Title: Role of high ubiquitin-conjugating enzyme E2 expression as a prognostic factor in nasopharyngeal carcinoma

doi: 10.3892/ol.2022.13314

Figure Lengend Snippet: UBE2B expression levels are a prognostic marker for patients with NPC receiving cisplatin-based chemoradiotherapy. Immunohistochemical analysis of UBE2B indicated nuclear and cytoplasmic staining in representative NPC cases with (A) low (H-score=125) and (B) high (H-score=375) expression (scale bars, 100 µm). (C) Survival analysis revealed that high expression of UBE2B was a prognostic marker for poor disease-specific survival, distal metastasis-free survival and local recurrence-free survival. NPC, nasopharyngeal carcinoma; UBE2B, ubiquitin-conjugating enzyme E2 B; Cum, cumulative.

Article Snippet: The slides were then washed with tris-buffered saline and incubated with a primary rabbit polyclonal antibody against UBE2B (1:100 dilution; GeneTex, Inc.) for 1 h. A ChemMate EnVision kit (DAKO; Agilent Technologies, Inc.) was applied to detect the primary antibody.

Techniques: Expressing, Marker, Immunohistochemical staining, Staining, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: Role of high ubiquitin-conjugating enzyme E2 expression as a prognostic factor in nasopharyngeal carcinoma

doi: 10.3892/ol.2022.13314

Figure Lengend Snippet: Associations between UBE2B expression levels and important clinicopathological variables.

Article Snippet: The slides were then washed with tris-buffered saline and incubated with a primary rabbit polyclonal antibody against UBE2B (1:100 dilution; GeneTex, Inc.) for 1 h. A ChemMate EnVision kit (DAKO; Agilent Technologies, Inc.) was applied to detect the primary antibody.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Role of high ubiquitin-conjugating enzyme E2 expression as a prognostic factor in nasopharyngeal carcinoma

doi: 10.3892/ol.2022.13314

Figure Lengend Snippet: Univariate log-rank analyses.

Article Snippet: The slides were then washed with tris-buffered saline and incubated with a primary rabbit polyclonal antibody against UBE2B (1:100 dilution; GeneTex, Inc.) for 1 h. A ChemMate EnVision kit (DAKO; Agilent Technologies, Inc.) was applied to detect the primary antibody.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Role of high ubiquitin-conjugating enzyme E2 expression as a prognostic factor in nasopharyngeal carcinoma

doi: 10.3892/ol.2022.13314

Figure Lengend Snippet: Multivariate survival analyses.

Article Snippet: The slides were then washed with tris-buffered saline and incubated with a primary rabbit polyclonal antibody against UBE2B (1:100 dilution; GeneTex, Inc.) for 1 h. A ChemMate EnVision kit (DAKO; Agilent Technologies, Inc.) was applied to detect the primary antibody.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Role of high ubiquitin-conjugating enzyme E2 expression as a prognostic factor in nasopharyngeal carcinoma

doi: 10.3892/ol.2022.13314

Figure Lengend Snippet: UBE2B modulates cisplatin cytotoxicity in nasopharyngeal carcinoma cells by targeting MGMT expression. (A) Western blot analysis demonstrated UBE2B and MGMT expression in TW01 cells with distinctive siRNA or plasmid transfection. (B) Cell viability assays were performed with TW01 cells to analyze the role of UBE2B and MGMT in cisplatin-induced cell death by using methylene blue staining. At least three independent experiments were performed. Cell survival results were presented as the mean ± standard deviation and compared using analysis of variance with Tukey's post-hoc test. *P<0.01, ***P<0.0001, 2BKD group vs. control group; # P<0.01, ### P<0.0001, 2BKD + MGMT group vs. 2BKD group. Ctrl, cells transfected with scrambled siRNA; 2BKD, cells transfected with siUBE2B; 2BKD + MGMT, cells transfected with siUBE2B plus pEGFPc1-MGMT. UBE2B, ubiquitin-conjugating enzyme E2 B; siRNA, small interfering RNA; MGMT, O6-methylguanine-DNA methyltransferase; pEGFP, plasmid expressing enhanced green fluorescence protein.

Article Snippet: The slides were then washed with tris-buffered saline and incubated with a primary rabbit polyclonal antibody against UBE2B (1:100 dilution; GeneTex, Inc.) for 1 h. A ChemMate EnVision kit (DAKO; Agilent Technologies, Inc.) was applied to detect the primary antibody.

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Staining, Standard Deviation, Control, Ubiquitin Proteomics, Small Interfering RNA, Fluorescence

Journal: Nature Communications

Article Title: In mouse chronic pancreatitis CD25 + FOXP3 + regulatory T cells control pancreatic fibrosis by suppression of the type 2 immune response

doi: 10.1038/s41467-022-32195-2

Figure Lengend Snippet: CP was induced in C57Bl/6 mice by repetitive caerulein injections over 4 weeks. In addition, animals received daily 30 µg of OC000459 or vehicle (0.625% DMSO). a Body weight changes over time were not significant between all groups (vehicle control n = 3/vehicle CP n = 8/OC000459 control n = 4/OC000459 CP n = 8). b OC000459 significantly reduced the CD4 + T cell infiltration of the pancreas in CP mice ( p = 0.0469, Veh. n = 8/OC n = 7), scale bars represent 20 µm. c The proportion of CD4 + STAT6 + cells was also smaller in CP tissue of OC000459 treated mice ( p = 0.0285, Veh. n = 8/OC n = 7). d , e Labeling of CD206 + showed a significant reduction of alternatively activated macrophages in the pancreas ( p = 0.0174, Veh. n = 8,/OC n = 7), scale bars represent 20 µm. d , f Immunofluorescence images showed a decreased labeling of collagen 1 and αSMA, whereas the amount of α-amylase in the pancreas of OC000459 treated mice was increased, scale bars represent 50 µm. g Quantification of immunofluorescence signals for αSMA + cells showed a significant decrease ( p = 0.0414, Veh. n = 7/OC n = 7), whereas the area of amylase + cells was significantly increased in the pancreas of OC000459 treated mice ( p = 0.0174, Veh. n = 8/OC n = 7). h Quantification by pattern quant software showed significantly less fibrotic tissue in OC000459 treated mice ( p = 0.0397, Veh. n = 8/OC n = 8). i H&E staining, azan blue staining and immune labeling of collagen 1 underlines the reduced fibrosis in the OC000459 treated group, scale bars represent 50 µm. j Gene expression analysis of pancreatic tissue by RT-qPCR demonstrated significantly decreased transcript levels for Mrc1 ( p = 0.0130, Veh. n = 6/OC n = 7), Col1a ( p = 0.0273, Veh. n = 7/OC n = 7), Il4 ( p = 0.0212, Veh. n = 6/OC n = 6), Il10 ( p = 0.0226, Veh. n = 6/OC n = 7), Areg ( p = 0.0104, Veh. n = 5/OC n = 6) and Tgfb ( p = 0.0433, Veh. n = 6/OC n = 7), indicating a reduced type 2 immune response. Transcript levels of Il13 (Veh. n = 5/OC n = 7) were reduced but the decrease did not reach significance. Transcript levels as determined by RT-qPCR were normalized using Rn5s as internal calibrator gene and were related to the corresponding mRNA amounts in control mice. All data were presented as means ± SEM, statistically significant differences were tested by unpaired two-tailed students t-test for independent samples and significance levels of p < 0.05 are marked by an asterisk ( c , e , h , g , j ). Source data are provided as a Source Data file.

Article Snippet: The CRTH2-specific inhibitor OC000459 (12027, Cayman Chemical) was injected i.p. once daily in a concentration of 30 μg in 1:160 dimethyl sulfoxide (A994.2, Roth).

Techniques: Control, Labeling, Immunofluorescence, Software, Staining, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 promotes a transcriptional downregulation of RAD51 and other proteins known to control DSB repair by HR. RNA-seq was performed on U2OS cells transfected with non-silencing (NS), NFKB2 or RELB siRNA. ( A ) Knockdown is efficient for both p100/52 and RelB, based on mRNA measurements by RNA-seq (left) and protein level by western blot (right). ( B ) KEGG pathway analysis of was performed to predict the 10 most upregulated and 10 most downregulated pathways in response to siNFKB2. Corresponding effects in response to siRELB are also displayed. ( C ) Expression levels are displayed for 39 genes with known relevance to HR, as defined by the KEGG database. Genes are arrayed from left to right based on degree of expression change after siNFKB2 (* denotes P < 0.01).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Control, RNA Sequencing, Transfection, Knockdown, Western Blot, Expressing

Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 promotes downregulation of RAD51 protein. ( A ) Representative western blots demonstrate that transcriptional silencing of key NF-κB proteins leads to reduced levels of RAD51 protein. ( B ) Quantitation of western blots from three independent experiments demonstrate that RAD51 reductions after siNFKB2 are reproducible (error bars denote the SEM). ( C ) Comparable levels of RAD51 knockdown are observed using three different siRNAs (each at 25 nM) targeting NFKB2 in U2OS cells. ( D ) Forced overexpression of the p52 fragment of Nfkb2 in mouse lung tissue is associated with increased Rad51 expression (error bars denote standard error, * denotes P < 0.02, NS denotes non-silencing control).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Western Blot, Quantitation Assay, Knockdown, Over Expression, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 inhibits DNA damage-induced RAD51 focus formation in human cancer cells. Cells previously transfected with siRNA or non-silencing (NS) controls were pulse-labeled with EdU to mark S-phase cells and concomitantly treated with 30 nM CPT for 1 hour. Cells were harvested and immune-stained as indicated. Representative microscopic images of unselected nuclei ( A ) are displayed for cells collected 3-hours after CPT treatment, and ( B ) quantitation of RAD51 foci per nucleus is plotted for each of the indicated conditions. ( C ) Quantitation of EdU intensity in CPT-untreated cells is used to estimate cell cycle at the time of the EdU pulse. ( D ) A schematic representation of the treatment timeline is displayed. ( E ) The mean number of RAD51 foci per EdU-positive nucleus is plotted as a function of time following CPT treatment (error bars denote SEM for at least 100 nuclei within a single experimental replicate, * denotes P < 0.05, *** denotes P < 1e−13, and ns denotes P > 0.05).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Transfection, Labeling, Staining, Quantitation Assay