Journal: Oncology Letters

Article Title: Tumor necrosis factor receptor 2 promotes growth of colorectal cancer via the PI3K/AKT signaling pathway

doi: 10.3892/ol.2016.5403

Figure Lengend Snippet: TNFR2 promoted Ki67 expression in CRC tissues and CRC cells. (A) TNFR2 and Ki67 expression in CRC tissues by IHC and statistical analysis. (B) TNFR2 and Ki67 expression in SW1116-NC-vector and SW1116-TNFR2-vector cells, and the quantitative histogram. (C) TNFR2 and Ki67 expression in HT29-NC-sh, HT29-TNFR2-sh1 and HT29-TNFR2-sh2 cells and the quantitative histogram. *P<0.05, **P<0.01. TNFR2, tumor necrosis factor receptor 2; CRC, colorectal cancer; IHC, immunohistochemistry; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Subsequent to being incubated at room temperature for 2 h in rabbit anti-human polyclonal primary antibodies against TNFR2 (catalog no. 19272-1-AP; dilution, 1:400; Proteintech Group Inc., Chicago, IL, USA) and Ki67 (working solution; catalog no. RB-9043; Maixin Biotech Co., Ltd., Fuzhou, China), the sections were washed with phosphate-buffered saline (PBS) and incubated in goat anti-rabbit/mouse IgG H&L (HRP) polymer (working solution) (catalog no. KIT-9921; Maixin Biotech Co., Ltd.) at room temperature for 30 min.

Techniques: Expressing, Plasmid Preparation, Immunohistochemistry

Journal: Oncology Letters

Article Title: Tumor necrosis factor receptor 2 promotes growth of colorectal cancer via the PI3K/AKT signaling pathway

doi: 10.3892/ol.2016.5403

Figure Lengend Snippet: TNFR2 promoted growth and proliferation of CRC cells. (A) Growth curves of SW1116-NC-vector and SW1116-TNFR2-vector cells. (B) Clone formation assay of SW1116-NC-vector and SW1116-TNFR2-vector, and quantitative histogram of clone numbers. (C) Growth curves of HT29-NC-sh, HT29-TNFR2-sh1 and HT29-TNFR2-sh2 cells. (D) Clone formation assay of HT29-NC-sh, HT29-TNFR2-sh1 and HT29-TNFR2-sh2 cells, and quantitative histogram of clone numbers. *P<0.05. TNFR2, tumor necrosis factor receptor 2; CRC, colorectal cancer; OD, optical density.

Article Snippet: Subsequent to being incubated at room temperature for 2 h in rabbit anti-human polyclonal primary antibodies against TNFR2 (catalog no. 19272-1-AP; dilution, 1:400; Proteintech Group Inc., Chicago, IL, USA) and Ki67 (working solution; catalog no. RB-9043; Maixin Biotech Co., Ltd., Fuzhou, China), the sections were washed with phosphate-buffered saline (PBS) and incubated in goat anti-rabbit/mouse IgG H&L (HRP) polymer (working solution) (catalog no. KIT-9921; Maixin Biotech Co., Ltd.) at room temperature for 30 min.

Techniques: Plasmid Preparation, Tube Formation Assay

Journal: Oncology Letters

Article Title: Tumor necrosis factor receptor 2 promotes growth of colorectal cancer via the PI3K/AKT signaling pathway

doi: 10.3892/ol.2016.5403

Figure Lengend Snippet: PI3K/AKT was required for the growth and proliferation of CRC cells induced by TNFR2. (A) pAKT, AKT, pERK, ERK expression in SW1116-NC-vector and SW1116-TNFR2-vector cells, as determined by western blot analysis. (B) pAKT, AKT, pERK, ERK expression in HT29-NC-sh, HT29-TNFR2-sh1 and HT29-TNFR2-sh2 cells, as determined by western blot analysis. (C) Ki67 and pAKT upregulation induced by TNFR2 in SW1116 cells was abrogated subsequent to treatment using LY294002. (D) Growth promotion of SW1116 cells induced by TNFR2 was inhibited following treatment using LY294002. (E) Clone formation promotion of SW1116 cells induced by TNFR2 was inhibited subsequent to treatment using LY294002 and quantitative histogram of clone numbers. *P<0.05. PI3K, phosphoinositide 3-kinase; (p)AKT, (phospho)-protein kinase B; (p)ERK, (phospho)-extracellular signal-related kinases; TNFR2, tumor necrosis factor receptor 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide; OD, optical density.

Article Snippet: Subsequent to being incubated at room temperature for 2 h in rabbit anti-human polyclonal primary antibodies against TNFR2 (catalog no. 19272-1-AP; dilution, 1:400; Proteintech Group Inc., Chicago, IL, USA) and Ki67 (working solution; catalog no. RB-9043; Maixin Biotech Co., Ltd., Fuzhou, China), the sections were washed with phosphate-buffered saline (PBS) and incubated in goat anti-rabbit/mouse IgG H&L (HRP) polymer (working solution) (catalog no. KIT-9921; Maixin Biotech Co., Ltd.) at room temperature for 30 min.

Techniques: Expressing, Plasmid Preparation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Proteomic study identifies Aurora-A–mediated regulation of alternative splicing through multiple splicing factors

doi: 10.1016/j.jbc.2024.108000

Figure Lengend Snippet: Aurora-A regulates splicing factor activity through phosphorylation. A , phosphorylation of SRSF3 by Aurora-A in vivo . Left panel: Phosphorylation of SRSF3 was analyzed upon Aurora-A inhibition in HeLa cells by Phos-tag gels on the top panel. The Western blot analysis of the SRSF3 is shown in the middle panel. TBP was used as a loading control as shown in the bottom panel. Right panel: Phosphorylation of SRSF3 was analyzed upon overexpression of WT or kinase-dead mutant (T287A, T288A) of Aurora-A in HeLa cells by Phos-tag gels. B , phosphorylation of SR proteins (SRSF1, SRSF3, and SRSF7) by Aurora-A was analyzed by autoradiography on the top panel. GST was used as a negative control. Coomassie staining of purified recombinant GST-tagged SR proteins and Western blot analysis of the HIS-tagged Aurora-A kinase are shown in the bottom two panels. The orange arrowhead indicates the auto-phosphorylation of Aurora-A and the green arrowhead indicates the phosphorylation of substrate (SR proteins). C , phosphorylation of hnRNP proteins (HNRNPC and PTBP1) by Aurora-A was analyzed by autoradiography on the top panel. GST was used as a negative control. Western blots of purified recombinant GST-tagged hnRNP proteins and HIS-tagged Aurora-A kinase are shown in the bottom two panels. The orange arrowhead indicates the auto-phosphorylation of Aurora-A and the green arrowhead indicates the phosphorylation of substrate (hnRNP proteins). GST was used as a negative control. D , RT-PCR assays monitoring endogenous splicing of CLK1 exon 4 in HEK293T upon inhibition of Aurora-A with MLN8237 (2 μM) for 72 h. Since the exon 4 skipped isoform undergoes rapid degradation by nonsense-mediated decay (NMD), cycloheximide (100 μg/ml) was applied for the last four hours to inhibit NMD and monitor splicing changes. PSI values are indicated. Two-tailed unpaired t-tests are applied. E , RT-PCR assays monitoring endogenous splicing of CLK1 exon 4 in HEK293T or HeLa cells upon knockdown of Aurora-A, SRSF3, or both for 72 h. Cycloheximide treatment was performed for the last 4 hours. PSI values are indicated. Two-tailed unpaired t-tests are applied.

Article Snippet: Two micrograms of total cell lysates were incubated with Dynabeads protein G (ThermoFisher Scientific #10004D) bound to 2μg of specific antibodies against SRSF1 (Proteintech, #12929-2-AP), SRSF3 (MBL, #RN080PW), SRSF7 (Proteintech, #11044-1-AP), TBP (Proteintech, 22006-1-AP), or Rabbit IgG (Proteintech, #30000-0-AP) overnight at 4 °C with rotation.

Techniques: Activity Assay, Phospho-proteomics, In Vivo, Inhibition, Western Blot, Control, Over Expression, Mutagenesis, Autoradiography, Negative Control, Staining, Purification, Recombinant, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Knockdown

Journal: Oncology Letters

Article Title: Identification of a novel FOXO3‑associated prognostic model in hepatocellular carcinoma

doi: 10.3892/ol.2025.14976

Figure Lengend Snippet: Identification of the 5-gene signature. (A) Forest plot of univariate Cox regression analysis in The Cancer Genome Atlas dataset. Cox results for the top five and bottom five genes. (B) Least absolute shrinkage and selection operator regression analysis. (C) λ curves show the least absolute shrinkage and the best λ was selected based on the minimum criteria. (D) The protein-protein interaction network between FOXO3 and 294 genes. FOXO3, DDX55, RAB10, RAB7A, TAF1B and TAF3 are highlighted by red dots and the remaining genes are represented by green dots. The color of the edges was determined by the combined score obtained from STRING. FOXO3, Forkhead box O3; DDX55, DEAD-box helicase 55; RAB10, RAB10, member RAS oncogene family; RAB7A, RAB7A, member RAS oncogene family; TAF1B, TATA-box binding protein associated factor, RNA polymerase I subunit B; TAF3, TATA-box binding protein associated factor 3; SLC4A1AP, solute carrier family 4 member 1 adaptor protein; ZNF765, zinc finger protein 765; MORC3, MORC family CW-type zinc finger 3; WWC2, WW and C2 domain containing 2; UBE3A, ubiquitin protein ligase E3A; SECISBP2L, SECIS binding protein 2 like; FAN1, FANCD2 And FANCI associated nuclease 1.

Article Snippet: The following antibodies were used: β-actin (1:1,000; cat. no. ET1702-52; HUABIO), DDX55 (1:1,000; cat. no. ER63225; HUABIO), RAB10 (1:1,000; cat. no. 11808-1-AP; Proteintech Group, Inc.), RAB7A (1:1,500; cat. no. 55469-1-AP; Proteintech Group, Inc.), TAF1B (1:500; cat. no. 12818-1-AP; Proteintech Group, Inc.), FOXO3 (1:1,000; cat. no. A9270; ABclonal Biotech Co., Ltd.) and TAF3 (1:500; cat. no. 18901-1-AP; Proteintech Group, Inc.), HRP conjugated goat anti-rabbit IgG polyclonal antibody (1:30000, HA1001, HUABIO), HRP conjugated goat anti-mouse IgG polyclonal antibody (1:30,000, HA1006, HUABIO).

Techniques: Selection, Binding Assay, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: Identification of a novel FOXO3‑associated prognostic model in hepatocellular carcinoma

doi: 10.3892/ol.2025.14976

Figure Lengend Snippet: Construction and validation of a 5-gene prognostic model. (A) The distribution of RS, survival status and expression of the 5-gene signature between the high- and low-risk groups in TCGA dataset. (B) Overall survival of patients with HCC in the high- and low- RS groups in TCGA dataset. (C) Time-dependent ROC curve of RS in TCGA dataset. (D) The distribution of RS, survival status and expression of the 5-gene signature between the high- and low-risk groups in the ICGC dataset. (E) Overall survival of patients with HCC in the high- and low-RS groups in the ICGC dataset. (F) Time-dependent ROC curve of RS in the ICGC dataset. RS, risk score; TCGA, The Cancer Genome Atlas; ICGC, International Cancer Genome Consortium; ROC, receiver operating characteristic; DDX55, DEAD-box helicase 55; RAB10, RAB10, member RAS oncogene family; RAB7A, RAB7A, member RAS oncogene family; TAF1B, TATA-box binding protein associated factor, RNA polymerase I subunit B; TAF3, TATA-box binding protein associated factor 3; AUC, area under the curve.

Article Snippet: The following antibodies were used: β-actin (1:1,000; cat. no. ET1702-52; HUABIO), DDX55 (1:1,000; cat. no. ER63225; HUABIO), RAB10 (1:1,000; cat. no. 11808-1-AP; Proteintech Group, Inc.), RAB7A (1:1,500; cat. no. 55469-1-AP; Proteintech Group, Inc.), TAF1B (1:500; cat. no. 12818-1-AP; Proteintech Group, Inc.), FOXO3 (1:1,000; cat. no. A9270; ABclonal Biotech Co., Ltd.) and TAF3 (1:500; cat. no. 18901-1-AP; Proteintech Group, Inc.), HRP conjugated goat anti-rabbit IgG polyclonal antibody (1:30000, HA1001, HUABIO), HRP conjugated goat anti-mouse IgG polyclonal antibody (1:30,000, HA1006, HUABIO).

Techniques: Biomarker Discovery, Expressing, Binding Assay

Journal: Oncology Letters

Article Title: Identification of a novel FOXO3‑associated prognostic model in hepatocellular carcinoma

doi: 10.3892/ol.2025.14976

Figure Lengend Snippet: Identification of five biomarkers in HCC. (A) mRNA expression of DDX55, RAB10, RAB7A, TAF1B and TAF3 in HCC and normal tissues from TCGA. Overall survival curves in patients with HCC with high or low expression of (B) DDX55, (C) RAB10, (D) RAB7A, (E) TAF1B and (F) TAF3 from TCGA dataset. The best cut-off value for each gene was obtained using the X-tile software. (G) mRNA expression of DDX55, RAB10, RAB7A, TAF1B and TAF3 in HCC and paired paracancerous tissues from 10 patients with HCC, assessed using reverse transcription-quantitative PCR. (H) Immunohistochemical images of DDX55, RAB10 and RAB7A in HCC and normal tissues obtained from the HPA. (I) Protein expression of DDX55, RAB10, RAB7A, TAF1B and TAF3 in HCC and paired paracancerous tissues from 10 patients with HCC, evaluated using western blot analysis. The figure on the right is a relative semi-quantitative histogram of protein expression. The gray values were obtained by ImageJ and analyzed using GraphPad Prism. (J) Cell Counting Kit-8 curves after knockdown of RAB10, RAB7A and TAF3 in Huh7 cells by siRNA. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. T, tumor; NT, not-tumor; HCC, hepatocellular carcinoma; TCGA, The Cancer Genome Atlas; DDX55, DEAD-box helicase 55; RAB10, RAB10, member RAS oncogene family; RAB7A, RAB7A, member RAS oncogene family; TAF1B, TATA-box binding protein associated factor, RNA polymerase I subunit B; TAF3, TATA-box binding protein associated factor 3; HPA, The Human Protein Atlas; NC, negative control.

Article Snippet: The following antibodies were used: β-actin (1:1,000; cat. no. ET1702-52; HUABIO), DDX55 (1:1,000; cat. no. ER63225; HUABIO), RAB10 (1:1,000; cat. no. 11808-1-AP; Proteintech Group, Inc.), RAB7A (1:1,500; cat. no. 55469-1-AP; Proteintech Group, Inc.), TAF1B (1:500; cat. no. 12818-1-AP; Proteintech Group, Inc.), FOXO3 (1:1,000; cat. no. A9270; ABclonal Biotech Co., Ltd.) and TAF3 (1:500; cat. no. 18901-1-AP; Proteintech Group, Inc.), HRP conjugated goat anti-rabbit IgG polyclonal antibody (1:30000, HA1001, HUABIO), HRP conjugated goat anti-mouse IgG polyclonal antibody (1:30,000, HA1006, HUABIO).

Techniques: Expressing, Software, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Western Blot, Cell Counting, Knockdown, Binding Assay, Negative Control

Journal: JCI Insight

Article Title: Cardiomyocyte d -dopachrome tautomerase protects against heart failure

doi: 10.1172/jci.insight.128900

Figure Lengend Snippet: (A) Cellular contractile function and calcium handling in cKO and CON mice 7 weeks after sham or TAC surgery. Isolated cardiomyocytes were electrically stimulated at 1 Hz. Sarcomere shortening, calcium transients, calcium uptake (+dR/dt), and release velocity (–dR/dt) were determined by Fura-2AM fluorescence in isolated cardiomyocytes. (B) Sarco-endoplasmic reticulum calcium ATPase (SERCA2a) expression in DDT-cKO mice 1 week after TAC. Immunoblots show SERCA2a content and NF-κB (p65 and p50) content in a nuclear (Nuc) extract of LV myocardium 1 week after TAC or sham surgery. GAPDH and TATA binding protein (TBP) were used as a cytoplasmic and a nuclear protein loading control, respectively. Data are shown as mean ± SEM; n = 5–7 mice per group. Significance determined by 1-way ANOVA with Tukey’s multiple-comparisons test. *P < 0.05; **P < 0.01; ****P < 0.0001 indicated by brackets. #P < 0.05 using 2-tailed Student’s t test to compare the differences between the sham and TAC in the cKO and CON groups.

Article Snippet: Nuclear and cytoplasmic extraction were performed with a kit protocol (Thermo Fisher Scientific, 78833) The antibodies used were directed against MIF ( 17 ), DDT ( 17 ), VEGF (Santa Cruz Biotechnology, sc-152), GAPDH (Cell Signaling Technology, 2118s), p-Akt T308 (CST, 4056s), Akt (CST, 4691), p-ERK (CST, 4370s), ERK (CST, 9102c), p–Smad-2 (CST, 3101), Smad-2 (CST, 5339), SERCA2a (Thermo Fisher Scientific, MA3-919) NF-κB p65 (CST, 8242), NF-κB p105/p50 (CST, 13586), and TBP (CST, 8515s).

Techniques: Isolation, Fluorescence, Expressing, Western Blot, Binding Assay

Journal: Neuron

Article Title: Antibody therapy targeting RAN proteins rescues C9 ALS/FTD phenotypes in C9orf72 mouse model

doi: 10.1016/j.neuron.2019.11.007

Figure Lengend Snippet: Antibodies

Article Snippet: Rabbit anti-PSMC4 , Proteintech , Cat#11389–1-AP;RRID:AB_2300373.

Techniques: Recombinant, Activity Assay, In Situ, Detection Assay, Software

Journal: Journal of Biological Chemistry

Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex

doi: 10.1074/jbc.ra118.006290

Figure Lengend Snippet: Figure 4. Nrf1 interacts with the TIP60 complex. (A) Wild-type (WT) and Nrf1-/- NIH-3T3 cell lines were treated with 200 nM carfilzomib (CFZ) for 8 hours. The cells were then subjected to chromatin immunoprecipitation (ChIP) with one of IgG, Nrf1, RUVBL1, or TIP60 antibodies. These samples were then analyzed by quantitative PCR with primers specific for antioxidant response element (ARE)- containing promoter regions of proteasome genes PSMA7, PSMB7, and PSMD12. Error bars denote SD (n=3). (B) HEK293 cells stably expressing tagged Nrf1 (Nrf13xFLAG) were treated or not with 200 nM CFZ for 8 hours. The cell lysates were then subjected to immunoprecipitation with anti-FLAG beads and analyzed by immunoblotting with antibodies specific for FLAG, RUVBL1, and RUVBL2. The lysate lanes were loaded with 5% of the input that was used for immunoprecipitation. The experiments were performed three independent times and a representative blot is shown.

Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Immunoprecipitation, Western Blot