Journal: Oncotarget

Article Title: FOXO4 expression is related to stem cell-like properties and resistance to treatment in diffuse large B-cell lymphoma

doi: 10.18632/oncotarget.13690

Figure Lengend Snippet: ( A ) Study scheme of gene expression analyses using doxorubicin (Dox) and phenylbutyrate (PB)-treated surviving cells: Four different cell lines (BJAB, Raji, Toledo, and Daudi) are treated with doxorubicin (300 nM) or phenylbutyrate (8 mM) for 48 h, and cDNA microarray analysis is done to identify differentially expressed target genes between treatment-surviving cells and parental control cells. ( B ) FOXO4 mRNA level is significantly higher in phenylbutyrate-treated surviving (PB) cells of BJAB, Raji and Daudi than control cells. ( C ) Vorinostat-treated surviving cells show higher mRNA level of FOXO4 than control (Con) cells of BJAB and Raji. ( D ) Primary lymphoma cells from three patients with refractory diffuse large B-cell lymphoma (DLBCL) show increased expression of FOXO4 in phenylbutyrate-treated surviving cells (PB) compared to the corresponding control cells. ( E ) The FOXO4 mRNA level is significantly higher in primary cells from four patients with refractory DLBCL than that of a patient with DLBCL who achieved complete response. Data represent means ± SD from three independent experiments.

Article Snippet: After endogenous peroxidase blocking for 5 min, tissues were incubated with primary polyclonal antibody against FOXO4 (1:1000; GTX50500; GeneTex, Irvine, CA, USA) for 15 min using a BOND-MAX autoimmunostainer (Leica Biosystems, Melbourne, Australia) for 15 min.

Techniques: Gene Expression, Microarray, Control, Expressing

Journal: Oncotarget

Article Title: FOXO4 expression is related to stem cell-like properties and resistance to treatment in diffuse large B-cell lymphoma

doi: 10.18632/oncotarget.13690

Figure Lengend Snippet: ( A, B ) Phenylbutyrate-treated surviving cells (BJAB-PB and Raji-PB) increase the expression of FOXO4 and its transcriptional targets (p21, p27, and SOD) compared to control cells. ( C, D ) BJAB-PB and Raji-PB cells show decreased expression of cyclin D1, CDK4, and cyclin A compared to control cells.

Article Snippet: After endogenous peroxidase blocking for 5 min, tissues were incubated with primary polyclonal antibody against FOXO4 (1:1000; GTX50500; GeneTex, Irvine, CA, USA) for 15 min using a BOND-MAX autoimmunostainer (Leica Biosystems, Melbourne, Australia) for 15 min.

Techniques: Expressing, Control

Journal: Oncotarget

Article Title: FOXO4 expression is related to stem cell-like properties and resistance to treatment in diffuse large B-cell lymphoma

doi: 10.18632/oncotarget.13690

Figure Lengend Snippet: ( A ) Soft agar colony formation in FOXO4-transfected or siFOXO4-transfected BJAB cells shows the amplification of FOXO4 increase colony forming ability and the knockdown of FOXO4 decrease colony formation in BJAB cell line. ( B ) Decrease in mRNA levels of Nanog, Oct-4 and Sox-2 in siFOXO4-transfected (siFOXO4) is noted compared to siControl-transfected (siCon) BJAB cells. ( C ) Phosphorylated AKT level is decreased in BJAB-PB cells with FOXO4 overexpression whereas siFOXO4-transfected BJAB-PB cells show the reverse of phosphorylated AKT. ( D ) The western blot shows the expression of FOXO4 protein in a BJAB clone (c2) overexpressing FOXO4. ( E ) Tumor sphere formation is observed from a BJAB clone (c2) overexpressing FOXO4. ( F ) Immunohistochemical staining for FOXO4 in tumor tissue of DLBCL (× 200). ( G ) Kaplan-Meier curves shows superior progression-free survival and overall survival of FOXO4-high group with diffuse large B cell lymphoma than FOXO4-low group. The P value is calculated using the log-rank test.

Article Snippet: After endogenous peroxidase blocking for 5 min, tissues were incubated with primary polyclonal antibody against FOXO4 (1:1000; GTX50500; GeneTex, Irvine, CA, USA) for 15 min using a BOND-MAX autoimmunostainer (Leica Biosystems, Melbourne, Australia) for 15 min.

Techniques: Transfection, Amplification, Knockdown, Over Expression, Western Blot, Expressing, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: FOXO4 expression is related to stem cell-like properties and resistance to treatment in diffuse large B-cell lymphoma

doi: 10.18632/oncotarget.13690

Figure Lengend Snippet: Characteristics of patients

Article Snippet: After endogenous peroxidase blocking for 5 min, tissues were incubated with primary polyclonal antibody against FOXO4 (1:1000; GTX50500; GeneTex, Irvine, CA, USA) for 15 min using a BOND-MAX autoimmunostainer (Leica Biosystems, Melbourne, Australia) for 15 min.

Techniques:

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