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: 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: Cells

Article Title: The Ebola Virus Nucleoprotein Recruits the Nuclear RNA Export Factor NXF1 into Inclusion Bodies to Facilitate Viral Protein Expression

doi: 10.3390/cells9010187

Figure Lengend Snippet: Interaction of NXF1 with nucleoprotein (NP) and VP35. ( A ) Immunoprecipitation of NXF1 and coIP of viral proteins. 293T cells were transfected with the plasmids encoding for flag/HA-NXF1 and one of the viral proteins NP, VP35, VP40 or VP30. Forty-eight hours p.t. flag/HA-NXF1 was precipitated using anti-flag antibodies, and input and precipitates were analyzed via SDS-PAGE and subsequent Western blot. NXF1 was detected with anti-flag antibodies, and anti-NP, anti-VP35, anti-VP40 or anti-VP30 antibodies were used for the detection of the respective viral protein. In case of the VP35 blot, the band at the lower edge of the blot that is partially cut off is caused by the light chain of the IP antibody. ( B ) Influence of p15 on the interaction between NXF1 and NP. Flag/HA-tagged NXF1 or p15 was precipitated from transfected 293T cells using anti-flag antibodies. Input and precipitate samples were subjected to SDS-PAGE and analyzed by Western blot using anti-flag antibodies for the detection of p15 and NXF1, whereas an anti-NP antibody was used for the detection of NP.

Article Snippet: Primary antibodies against NP (rabbit anti-EBOV NP pAb, cat. no. 0301-012), VP35 (rabbit anti-ZEBOV VP35 affinity purified polyclonal antibody, cat. no. 0301-040) and VP40 (mouse anti-EBOV VP40 mAb, clone 3G5, cat. no. 0201-016) were obtained from IBT Bioservices, and the antibody against NXF1 (mouse anti-NXF1 mAb, clone 53H8, cat. no. ab50609) was obtained from Abcam.

Techniques: Immunoprecipitation, Transfection, SDS Page, Western Blot

Journal: Cells

Article Title: The Ebola Virus Nucleoprotein Recruits the Nuclear RNA Export Factor NXF1 into Inclusion Bodies to Facilitate Viral Protein Expression

doi: 10.3390/cells9010187

Figure Lengend Snippet: Influence of NXF1 on viral RNA synthesis and protein expression and interaction of NXF1 with viral RNAs. ( A ) Analysis of viral RNA synthesis and protein expression in NXF1 knockdown cells. 293T cells were transfected with siRNAs targeting either NXF1 (anti-NXF1) or L (anti-L) or with a negative control (ctrl) siRNA. Forty-eight hours p.t., cells were transfected with the plasmids required for a replication-competent minigenome assay. Forty-eight hours later, reporter activity was measured. ( B ) Analysis of viral transcription and protein expression in NXF1 knockdown cells in the absence of genome replication. 293T cells were transfected with siRNAs and 48 h p.t. with the plasmids required for a replication-deficient minigenome assay. After a further 48 h, reporter activity was analyzed. ( C ) Analysis of viral mRNA levels in NXF1 knockdown cells. Forty-eight hours after siRNA transfection of 293T cells, the cells were transfected with all components for the replication-competent minigenome assay. Forty-eight hours later RNA was isolated and RT-qPCR for viral mRNA was performed. ( D ) Analysis of viral gene expression after infection of NXF1 knockdown cells infected with recombinant EBOV expressing firefly luciferase. Reporter activity was determined one day p.i. and is shown relative to the reporter activity of cells treated with negative control siRNA. ( E ) Influence of NXF1 knockdown on viral mRNA synthesis. RNA from cells from panel D was isolated and luciferase mRNA was quantified using RT-qPCR. ( F ) Interaction of NXF1 with viral mRNA. 293T cells were transfected with plasmids encoding flag/HA-NXF1 or flag/HA-NXF1-mutants as indicated, in addition to the replication-competent minigenome, T7-polymerase, and the RNP proteins. As a control L was omitted (-L). Further negative controls (shown in grey) were as indicated. Forty-eight hours p.t., pulldowns were performed using anti-flag antibodies and RNA was isolated from the precipitates. mRNA levels in the precipitates were analyzed by subsequent RT-qPCR. ( G ) Interaction of NXF1 with viral genomic RNA. IP with anti-flag antibodies was performed with cell lysates from 293T cells transfected with plasmids encoding the replication-competent minigenome, T7-polymerase, and either flag/HA-NXF1, or flag/HA-NP, VP35, and VP24 (RNP). In controls the minigenome was omitted (-1cis). Further negative controls (shown in grey) were as indicated. RNA was isolated from the precipitates and analyzed via RT-qPCR. Means and standard deviations for two independent experiments (six biological replicates) are shown in panels D and E , and for three independent experiments in all other panels. Asterisks indicate p values from one-way ANOVA (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001; ns: p > 0.05).

Article Snippet: Primary antibodies against NP (rabbit anti-EBOV NP pAb, cat. no. 0301-012), VP35 (rabbit anti-ZEBOV VP35 affinity purified polyclonal antibody, cat. no. 0301-040) and VP40 (mouse anti-EBOV VP40 mAb, clone 3G5, cat. no. 0201-016) were obtained from IBT Bioservices, and the antibody against NXF1 (mouse anti-NXF1 mAb, clone 53H8, cat. no. ab50609) was obtained from Abcam.

Techniques: Expressing, Knockdown, Transfection, Negative Control, Activity Assay, Isolation, Quantitative RT-PCR, Gene Expression, Infection, Recombinant, Luciferase, Control