Journal: Molecular Cancer

Article Title: Identification of cyclin B1 and Sec62 as biomarkers for recurrence in patients with HBV-related hepatocellular carcinoma after surgical resection

doi: 10.1186/1476-4598-11-39

Figure Lengend Snippet: GO and pathway ehrichment and interaction network analyses based on the set of 615 differentially expressed genes in the recurrent HCC and non-recurrent HCC samples . A. GO category based on the biological process for differentially expressed genes. (Upper) The significant GO category for the upregulated genes. (Below) The significant GO category for downregulated genes. LgP is the base-10 logarithm of the p value. B. KEGG pathway analysis for the differentially expressed genes. (Upper) The significant pathway for the upregulated genes. (Below) The significant pathway for the downregulated genes. C. Interaction network analysis of the 615 genes. The 615 altered genes were connected in a network based on prior known protein-protein interactions and signaling pathways. Blue, upregulated genes. Red, downregulated genes. The CCNB1 , SEC62 and BIRC3 genes had the highest DiffK ( i ) values; therefore, they might be of great importance to HCC recurrence in these patients.

Article Snippet: Cyclin B1 (V152) mouse mAb and Birc3 (58 C7) rabbit mAb were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Protein-Protein interactions

Journal: Molecular Cancer

Article Title: Identification of cyclin B1 and Sec62 as biomarkers for recurrence in patients with HBV-related hepatocellular carcinoma after surgical resection

doi: 10.1186/1476-4598-11-39

Figure Lengend Snippet: High expression levels of cyclin B1, Sec62, and Birc3 in the patients with recurrent HCC. A. The mRNA (left) and protein (right) expression of cyclin B1 was significantly higher in the patients with recurrent HCC than in those with non-recurrent HCC and in the healthy controls ( p < 0.001). However, no difference was found between the non-recurrence and healthy groups ( p > 0.05). B. The mRNA (left) and protein (right) expression of Sec62 were significantly higher in the patients with recurrent HCC than those with non-recurrent HCC and in the healthy controls ( p < 0.001). However, no difference was found between the non-recurrence and healthy groups ( p > 0.05). C. The mRNA (left) and protein (right) expression levels of Birc3 were significantly higher in the patients with recurrent HCC than in those with non-recurrent HCC and in the healthy controls ( p < 0.001). However, no difference was found between the non-recurrence and healthy groups ( p > 0.05). * Compared with non-recurrence group. # Compared with the healthy group. The mRNA expression levels were quantified using quantitative PCR. GAPDH was used as the endogenous control for the mRNA levels. The protein levels were examined by western blotting. β-actin was used as the endogenous control for the protein levels.

Article Snippet: Cyclin B1 (V152) mouse mAb and Birc3 (58 C7) rabbit mAb were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Molecular Cancer

Article Title: Identification of cyclin B1 and Sec62 as biomarkers for recurrence in patients with HBV-related hepatocellular carcinoma after surgical resection

doi: 10.1186/1476-4598-11-39

Figure Lengend Snippet: High expression levels of cyclin B1, Sec62, and Birc3 in the livers of the patients with recurrent HCC. A. Immunohistochemestry stain for cyclin B1, Sec62, and Birc3 expression (original magnification × 400). Strong expression of these proteins was obserced in the livers of the patients with recurrent HCC. By contrast, the expression of these proteins was apparently decreased in the patients with non-recurrent HCC. B. cyclin B1, Sec62, and Birc3 immunohistochemical indices. The cyclin B1, Sec62, and Birc3 immunohistochemical indices were significantly higher in the recurrent HCC samples than in the non-recurrent HCC samples. ( p < 0.05). The data are mean ± SD values. * p < 0.05 compared with the non-recurrence group.

Article Snippet: Cyclin B1 (V152) mouse mAb and Birc3 (58 C7) rabbit mAb were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: Molecular Cancer

Article Title: Identification of cyclin B1 and Sec62 as biomarkers for recurrence in patients with HBV-related hepatocellular carcinoma after surgical resection

doi: 10.1186/1476-4598-11-39

Figure Lengend Snippet: The clinical significance of cyclin B1, Sec62, and Birc3 expressions in the patients with HCC surgery. Kaplan-Meier curves were used to estimate the recurrence-free survival rates according to the expression levels of cyclin B1, Sec62, and Birc3 in the 80 HCC patients who underwent surgery. Cyclin B1 (left), Sec62 (middle), and Birc3 (right).

Article Snippet: Cyclin B1 (V152) mouse mAb and Birc3 (58 C7) rabbit mAb were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing

Journal: Molecular Cancer

Article Title: Identification of cyclin B1 and Sec62 as biomarkers for recurrence in patients with HBV-related hepatocellular carcinoma after surgical resection

doi: 10.1186/1476-4598-11-39

Figure Lengend Snippet: Univariate analyses of predictors of recurrence in HCC patients

Article Snippet: Cyclin B1 (V152) mouse mAb and Birc3 (58 C7) rabbit mAb were purchased from Cell Signaling Technology (Danvers, MA).

Techniques:

Journal: Scientific reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats.

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: Figure 2. BIRC3 expression segregates mesenchymal GBM from the other GBM subtypes. Normalized log2 expression was compared across the neural, proneural, classical, and mesenchymal GBM subtypes. Results are represented in box plot format: (A) BIRC3; (B). BIRC2; (C) BIRC5; (D) NF1; (E) ZEB1; and (F) CREB1.

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Expressing

Journal: Scientific reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats.

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: Figure 3. BIRC3 expression is a unique identifier of mesenchymal GBM. Normalized log2 expression of BIRC3 was plotted against other proposed mesenchmymal-selective genes in a scatter plot, colored by GBM subtypes and with r representing Pearsons correlation coefficient. (A) BIRC3 versus CREB1 expression. (B) BIRC3 versus NF1 expression. (C) BIRC3 versus ZEB1 expression.

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Expressing

Journal: Scientific reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats.

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: Figure 4. BIRC3 is expressed at higher level in the tumor cell niche compared to the vascular niche in GBM. Human GBM tissue microarray was stained for BIRC3 (Brown). BIRC3 expression can be compared between GBM tumor cell niche and vascular endothelial cell niche (A,B). Please note the focus of microvascular proliferation which shows negligible BIRC3 expression (Red arrow).

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Microarray, Staining, Expressing

Journal: Scientific reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats.

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: Figure 5. Hypoxia induces BIRC3 expression in GBM ex vivo and in vivo. (A) U87 GBM cells were cultured under hypoxic conditions (1% O2) for 24 hr and BIRC3 gene expression was analyzed by RT-PCR. Data are representative of three independent experiments (p < 0.05). (B) U87 cells were cultured under hypoxia conditions (1% O2) for the indicated intervals and BIRC3 protein levels were determined by western blot. Data are representative of three independent experiments. Whole images for Western-blot can be found in the Supplementary Figure 7. (C–E) GBM xenografts were established by by injecting 2 × 106 U87 cells on the flank of 6–8 week nude mice. At 6 weeks, mice were sacrificed and xenografts were assessed for BIRC3 (brown) and CA9 (pink) or HIF-1α (pink) expression by IHC. (C) BIRC3 and CA9 expression. (D) HIF-1α expression. (E) BIRC3 and HIF-1α expression.

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Expressing, Ex Vivo, In Vivo, Cell Culture, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Scientific reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats.

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: Figure 6. Inhibition of HIF-1α blocks hypoxia-induced up-regulation of BIRC3 expression in GBM. (A,B) U87 GBM cells, or these cells transfected with HIF-1α siRNA for 48 hr, were cultured under hypoxia conditions (1% O2) for 24 hr. Efficiency of HIF-1α knockdown and effects on BIRC3 gene expression were determined by qRT-PCR (n = 3 independent experiments, p < 0.05). (C) BIRC3 protein level were also assessed following knockdown of HIF-1α + /− hypoxia (n = 3 independent experiments). Whole images for Western- blot can be found in the Supplementary Figure 8. (D) ChIP of HIF-1α on the BIRC3 gene promoter was performed in U87 GBM cells exposed to hypoxia (1% O2 for 24 hr).

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Inhibition, Expressing, Transfection, Cell Culture, Knockdown, Gene Expression, Quantitative RT-PCR, Western Blot

Journal: Scientific reports

Article Title: BIRC3 is a biomarker of mesenchymal habitat of glioblastoma, and a mediator of survival adaptation in hypoxia-driven glioblastoma habitats.

doi: 10.1038/s41598-017-09503-8

Figure Lengend Snippet: Figure 7. BIRC3 silencing impairs hypoxia-induced survival of GBM to radiotherapy (RT). 1 × 104 U87 MG GBM cells were cultured in 96 well plate under hypoxia (1% O2) condition for 12 hr and irradiated with 4 Gy. Cells were returned to hypoxia conditions for another 12 hr and harvested. BIRC3 mRNA and protein expression were analyzed by RT-PCR (A) and Western blot, respectively (B). Similar results were obtained from three independent experiments (p < 0.05). Whole images for Western-blot can be found in the Supplementary Figure 9. (C) U87 GBM cells with or without BIRC3 siRNA pretreatment (48 hr earlier) were cultured under hypoxia (1% O2) for 12 hr and irradiated with 2 Gy, 4 Gy, 6 Gy or 8 Gy. Cells were returned to hypoxia conditions for another 24 hr and cell survival were assessed using an XTT Cell Viability Assay Kit. Data are representative of three independent experiments (p < 0.05).

Article Snippet: Antibodies used were mouse anti-human BIRC3 (R&D Systems MAB817, 1:600) and rabbit anti-human CA9 (Novus Biologicals NB100–417SS, 1:1500).

Techniques: Cell Culture, Irradiation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Viability Assay

Journal: Oncotarget

Article Title: Brassinin inhibits STAT3 signaling pathway through modulation of PIAS-3 and SOCS-3 expression and sensitizes human lung cancer xenograft in nude mice to paclitaxel

doi:

Figure Lengend Snippet: (A) Cell proliferation assay was performed using the Roche xCELLigence Real-Time Cell Analyzer (RTCA) DP instrument (Roche Diagnostics GmbH, Germany) as described under “Material and methods”. A549 cells (5 × 10 3 cells/well) were seeded onto 16-well E-plates and continuously monitored using impedance technology. (B) Invasion assay was performed using the Roche xCELLigence Real-Time Cell Analyzer (RTCA) DP instrument (Roche Diagnostics GmbH, Germany) as described under ‘Materials and methods’. We tested the effect of BSN on A549 cell invasive activity (4 × 10 4 cells/well) in the matrigel-coated CIM (cellular invasion/migration)-plate 16 exposed to various indicated concentrations of BSN. (C) A549 cells (1 × 10 6 cells/well) were seeded onto 6-well plates, they were treated with 300 μM of BSN for 24 h. Then, the cells were harvested, washed with a cold PBS buffer, and digested with RNase A. Cellular DNA staining with propidium iodide and flow cytometric analysis was done to determine the cell cycle distribution as described in “Materials and methods”. (D) A549 cells (1 × 10 6 cells/well) were treated with 300 μM of BSN for 24 h. The cells were incubated with an FITC-conjugated annexin V antibody and then analyzed by a flow cytometry as described in “Materials and methods”. (E) A549 cells (1 × 10 6 cells/well) were treated with indicated concentrations of BSN, after which whole-cell extracts were prepared and 20 μg portions of those extracts were resolved on 10% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed against caspase-3 and PARP antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (F) A549 cells (1 × 10 6 cells/well) were treated with indicated concentrations of BSN, after which whole-cell extracts were prepared and 20 μg portions of those extracts were resolved on 10% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed against Bcl-xl, Bcl-2, Survivin, Cyclin D1, IAP1, IAP2, VEGF, MMP-9, and COX-2 antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown here are representative of three independent experiments.

Article Snippet: Rabbit polyclonal antibody against STAT3 and mouse monoclonal antibodies against phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), Bcl-2, Bcl-xl, PIAS-3, SOCS-3, Caspase-3, Cyclin D1, IAP1, IAP2, COX-2, MMP-9, Survivin, VEGF, PARP, phospho-Akt (Ser473), Akt, Ki-67, CD31, β-actin, goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Proliferation Assay, Invasion Assay, Activity Assay, Migration, Staining, Incubation, Flow Cytometry, SDS Page

Journal: Oncotarget

Article Title: Brassinin inhibits STAT3 signaling pathway through modulation of PIAS-3 and SOCS-3 expression and sensitizes human lung cancer xenograft in nude mice to paclitaxel

doi:

Figure Lengend Snippet: (A) A549 cells (1× 10 4 cells/well) were treated with BSN (0, 25, 50, and 75 μM), and paclitaxel (0, 1, 2.5, and 5 nM) for 24 h. The cytotoxicity was determined by MTT assays ( left ). BSN synergistically enhances paclitaxel-induced cell death in A549 cells ( right) . The average of the CI values obtained at nine different combinations. CI of less than 1 was considered synergistic; CI of 1 was considered additive and a CI greater than 1 antagonistic. (B) A549 cells (1 × 10 6 cells/well) were co-treated with 25 μM of BSN and 1 nM of paclitaxel for 24 h. After which whole-cell extracts were prepared and 20 μg portions of those extracts were resolved on 10% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed against Bcl-xl, Bcl-2, Cyclin D1, Survivin, and IAP1 antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown here are representative of three independent experiments. (C) A549 cells (1 × 10 6 cells/well) were co-treated with 25 μM of BSN and 1 nM paclitaxel for 24 h. The cells were incubated with an FITC-conjugated annexin V antibody and then analyzed by a flow cytometry as described in “Materials and methods”. (D) A549 cells (1 × 10 6 cells/well) were co-treated with 25 μM of BSN and 1 nM paclitaxel for 24 h. After which whole-cell extracts were prepared and 20 μg portions of those extracts were resolved on 10% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed against caspase-3 and PARP antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown here are representative of three independent experiments.

Article Snippet: Rabbit polyclonal antibody against STAT3 and mouse monoclonal antibodies against phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), Bcl-2, Bcl-xl, PIAS-3, SOCS-3, Caspase-3, Cyclin D1, IAP1, IAP2, COX-2, MMP-9, Survivin, VEGF, PARP, phospho-Akt (Ser473), Akt, Ki-67, CD31, β-actin, goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: SDS Page, Incubation, Flow Cytometry

Journal: Oncotarget

Article Title: Brassinin inhibits STAT3 signaling pathway through modulation of PIAS-3 and SOCS-3 expression and sensitizes human lung cancer xenograft in nude mice to paclitaxel

doi:

Figure Lengend Snippet: (A) Immunohistochemical analysis of phospho-STAT3 showed the inhibition inexpression of phospho-STAT3 in BSN either alone or in combination with paclitaxel-treated samples, as compared with control group (upper panels). Percentage indicates positive staining for the given biomarker. The photographs were taken at the magnification of 40. Immunohistochemical analysis of proliferation marker Ki-67 indicates the inhibition of lung cancer cell proliferation in BSN either alone or in combination with paclitaxel-treated groups of animals (middle panels). Immunohistochemical analysis of CD31 for microvessel density in lung cancer tumors indicates the inhibition of angiogenesis by either BSN alone and in combination with paclitaxel (lower panels). (B) Western blot analysis showed the inhibition of phospho-STAT3 (Tyr705), phospho-JAK1 (Tyr1022/1023), phospho-JAK2 (Tyr1007/1008), phospho-Src (Tyr416), and PIAS-3 by BSN either alone or in combination with paclitaxel-treated groups in whole cell extracts from mice tissue. Western samples from three mice in each group were analyzed and representative data are shown. (C) Equal amounts of lysates were analyzed by Western blot analysis using antibodies against cell survival (Bcl-xl, Bcl-2, and Survivin), proliferation (Cyclin D1), anti-apoptotic (IAP1). Western samples from three mice in each group were analyzed and representative data are shown. (D) Equal amounts of lysates were analyzed by Western blot analysis using antibodies against Procaspase-3, cleaved caspase-3, PARP, and cleaved PARP. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. Western samples from three mice in each group were analyzed and representative data are shown.

Article Snippet: Rabbit polyclonal antibody against STAT3 and mouse monoclonal antibodies against phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), Bcl-2, Bcl-xl, PIAS-3, SOCS-3, Caspase-3, Cyclin D1, IAP1, IAP2, COX-2, MMP-9, Survivin, VEGF, PARP, phospho-Akt (Ser473), Akt, Ki-67, CD31, β-actin, goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunohistochemical staining, Inhibition, Control, Staining, Biomarker Discovery, Marker, Western Blot

Journal: World Journal of Gastroenterology

Article Title: Human antigen R mediated post-transcriptional regulation of inhibitors of apoptosis proteins in pancreatic cancer

doi: 10.3748/wjg.v25.i2.205

Figure Lengend Snippet: Pancreatic cancer specimens displayed increased Inhibitors of apoptosis proteins expression analysis in cancer tissues. mRNA and protein expression of inhibitors of apoptosis proteins in normal tissues ( n = 9) and pancreatic cancer ( n = 61) were evaluated by A: quantitative reverse transcription polymerase chain reaction; and B: western blot analysis. IAP1: Inhibitor of apoptosis protein 1; IAP2: Inhibitor of apoptosis protein 2; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The following primary antibodies were used: 1:1000 mouse monoclonal anti-HuR from Abcam (ab186430), 1:5000 rabbit monoclonal anti-IAP1 from Abcam (ab108361), 1:1000 rabbit monoclonal anti-IAP2 from Abcam (ab32059), and 1:10000 mouse monoclonal anti-GAPDH from Ambion (AM4300).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: World Journal of Gastroenterology

Article Title: Human antigen R mediated post-transcriptional regulation of inhibitors of apoptosis proteins in pancreatic cancer

doi: 10.3748/wjg.v25.i2.205

Figure Lengend Snippet: Univariate analysis of histopathological features (log-rank)

Article Snippet: The following primary antibodies were used: 1:1000 mouse monoclonal anti-HuR from Abcam (ab186430), 1:5000 rabbit monoclonal anti-IAP1 from Abcam (ab108361), 1:1000 rabbit monoclonal anti-IAP2 from Abcam (ab32059), and 1:10000 mouse monoclonal anti-GAPDH from Ambion (AM4300).

Techniques: Expressing

Journal: World Journal of Gastroenterology

Article Title: Human antigen R mediated post-transcriptional regulation of inhibitors of apoptosis proteins in pancreatic cancer

doi: 10.3748/wjg.v25.i2.205

Figure Lengend Snippet: Multivariate analysis for overall survival

Article Snippet: The following primary antibodies were used: 1:1000 mouse monoclonal anti-HuR from Abcam (ab186430), 1:5000 rabbit monoclonal anti-IAP1 from Abcam (ab108361), 1:1000 rabbit monoclonal anti-IAP2 from Abcam (ab32059), and 1:10000 mouse monoclonal anti-GAPDH from Ambion (AM4300).

Techniques: Expressing

Journal: World Journal of Gastroenterology

Article Title: Human antigen R mediated post-transcriptional regulation of inhibitors of apoptosis proteins in pancreatic cancer

doi: 10.3748/wjg.v25.i2.205

Figure Lengend Snippet: Survival analysis. A: The survival time of patients with the low expression of inhibitor of apoptosis protein 1 tended to be longer than those with high expression ( P < 0.05). B: There was no difference of survival among high and low inhibitor of apoptosis protein 2 (IAP2) mRNA expression. IAP1: Inhibitor of apoptosis protein 1; IAP2: Inhibitor of apoptosis protein 2.

Article Snippet: The following primary antibodies were used: 1:1000 mouse monoclonal anti-HuR from Abcam (ab186430), 1:5000 rabbit monoclonal anti-IAP1 from Abcam (ab108361), 1:1000 rabbit monoclonal anti-IAP2 from Abcam (ab32059), and 1:10000 mouse monoclonal anti-GAPDH from Ambion (AM4300).

Techniques: Expressing

Journal: World Journal of Gastroenterology

Article Title: Human antigen R mediated post-transcriptional regulation of inhibitors of apoptosis proteins in pancreatic cancer

doi: 10.3748/wjg.v25.i2.205

Figure Lengend Snippet: Pancreatic cancer specimens displayed increased human antigen R expression analysis in cancer tissues. mRNA and protein expression of human antigen R (HuR) were upregulated by A: quantitative reverse transcription polymerase chain reaction; and B: western blot analysis. C: Immunohistochemistry showed that HuR was mainly positive in the ductal cancer cell’s nucleus and less in cytoplasm. D: Expressions of inhibitors of apoptosis proteins were correlated with HuR expression. IAP1: Inhibitor of apoptosis protein 1; IAP2: Inhibitor of apoptosis protein 2; HuR: Human antigen R; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PDAC: Pancreatic ductal adenocarcinoma.

Article Snippet: The following primary antibodies were used: 1:1000 mouse monoclonal anti-HuR from Abcam (ab186430), 1:5000 rabbit monoclonal anti-IAP1 from Abcam (ab108361), 1:1000 rabbit monoclonal anti-IAP2 from Abcam (ab32059), and 1:10000 mouse monoclonal anti-GAPDH from Ambion (AM4300).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry

Journal: World Journal of Gastroenterology

Article Title: Human antigen R mediated post-transcriptional regulation of inhibitors of apoptosis proteins in pancreatic cancer

doi: 10.3748/wjg.v25.i2.205

Figure Lengend Snippet: Human antigen R silencing is associated with inhibitors of apoptosis proteins mRNA and protein expression regulation in pancreatic cancer cells. Human antigen R (HuR) silencing decreased inhibitor of apoptosis protein 1 (IAP1) and increased inhibitors of apoptosis protein 2 (IAP2) mRNA expression (A) and decreased IAP1 and increased IAP2 protein expression (B). IAP1: Inhibitor of apoptosis protein 1; IAP2: Inhibitor of apoptosis protein 2; HuR: Human antigen R; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The following primary antibodies were used: 1:1000 mouse monoclonal anti-HuR from Abcam (ab186430), 1:5000 rabbit monoclonal anti-IAP1 from Abcam (ab108361), 1:1000 rabbit monoclonal anti-IAP2 from Abcam (ab32059), and 1:10000 mouse monoclonal anti-GAPDH from Ambion (AM4300).

Techniques: Expressing

Journal: Oncotarget

Article Title: Long non-coding RNA CRNDE promotes gallbladder carcinoma carcinogenesis and as a scaffold of DMBT1 and C-IAP1 complexes to activating PI3K-AKT pathway

doi: 10.18632/oncotarget.12023

Figure Lengend Snippet: ( A – B ) Heat map for the Relative expression of si-DMBT1 and si-NC in GBC-SD cells analyzed by using GSEA datasets and bioinformatics predictions. ( C ) RIP analyses were performed using antibodies against DMBT1, with IgG as a negative control in GBC-SD cells. The enrichment of the CRNDE was detected using RT-PCR and normalized to the input. ( D ) RNA pull-down was performed using a CRNDE template and RNA-binding protein separated by SDS-PAGE in GBC-SD cells. The protein bands were excised and detected by mass spectrometry analysis. ( E – F ) C-IAP1 was detected by the Western blotting assay in the samples pulled down by CRNDE. RIP analyses were performed using antibodies against c-IAP1, with IgG as a negative control in GBC-SD cells. The enrichment of the CRNDE was detected using RT-PCR and normalized to the input. ( G – H ) Co-IP assay detected by western blot. IgG as a negative control in GBC-SD cells.

Article Snippet: Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted with antibodies against Rabbit anti-human DMBT1 antibody and rabbit anti-humanC-IAP1 antibody (Cell Signaling Technology, CA, USA); antibodies of PI3K-AKT pathway which include: MMP-9, JUK, P-JUK, ERK, P-ERK, AKT, P-AKT(Cell Signaling Technology, CA, USA); normal mouse or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FLAG, mouse anti-G FP (Sigma, CA, USA).

Techniques: Expressing, Negative Control, Reverse Transcription Polymerase Chain Reaction, RNA Binding Assay, SDS Page, Mass Spectrometry, Western Blot, Co-Immunoprecipitation Assay

Journal: Oncotarget

Article Title: Long non-coding RNA CRNDE promotes gallbladder carcinoma carcinogenesis and as a scaffold of DMBT1 and C-IAP1 complexes to activating PI3K-AKT pathway

doi: 10.18632/oncotarget.12023

Figure Lengend Snippet: ( A ) Deletion mapping of the DMBT1 domain in binding with CRNDE. RIP assays show association of deletion-DMBT1 domain with CRNDE in GBC-SD cells. Relative enrichment (means ± s.e.m.) represents RNA levels associated with domains relative to an input control from three independent experiments. Antibodies against FLAG and control IgG served as controls. ( B ) GBS-SD cells with stable overexpression of DMBT1 or knockdown of DMBT1 were treated with Actinomycin D (2.5 μM) for 24 h. Detect the mRNA level by RT-PCR. ( C ) Migration numbers of transwell assay of different deletion mapping of the DMBT1 domains. ( D ) Deletion mapping of the C-IAP1-binding domain in CRNDE. Top, diagrams of full-length CRNDE and the deletion fragments. Middle, the in vitro –transcribed full-length CRNDE and deletion fragments show the correct sizes. Bottom, immunoblot analysis for C-IAP1 in protein samples pulled down by the different CRNDE constructs. ( E ) Migration numbers of transwell assay of different deletion mapping of the CRNDE fragments. ( F ) GBS-SD cells with stable overexpression of CRNDE or knockdown of CRNDE were treated with MG132 (5 μM) for 24 h. Cell lysates were immunoprecipitated with antibody against C-IAP1. The precipitates and input were analyzed by immunoblotting.

Article Snippet: Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted with antibodies against Rabbit anti-human DMBT1 antibody and rabbit anti-humanC-IAP1 antibody (Cell Signaling Technology, CA, USA); antibodies of PI3K-AKT pathway which include: MMP-9, JUK, P-JUK, ERK, P-ERK, AKT, P-AKT(Cell Signaling Technology, CA, USA); normal mouse or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FLAG, mouse anti-G FP (Sigma, CA, USA).

Techniques: Binding Assay, Control, Over Expression, Knockdown, Reverse Transcription Polymerase Chain Reaction, Migration, Transwell Assay, In Vitro, Western Blot, Construct, Immunoprecipitation

Journal: Oncotarget

Article Title: Long non-coding RNA CRNDE promotes gallbladder carcinoma carcinogenesis and as a scaffold of DMBT1 and C-IAP1 complexes to activating PI3K-AKT pathway

doi: 10.18632/oncotarget.12023

Figure Lengend Snippet: ( A ) RT-qPCR and western blot were used to detect the mRNA levels of CRNDE and protein level of c-IAP1 in knockdown and overexpression DMBT1 cells. ( B ) RT-qPCR was used to detect the mRNA levels of CRNDE and c-IAP1 in knockdown and overexpression DMBT1 cells. ( C ) RT-qPCR was used to detect the mRNA levels of CRNDE and c-IAP1 in knockdown and overexpression DMBT1 cells when knockdown of CRNDE. ( D ) Validation of the RNA-seq results in GBC-SD cells using qRT-PCR. A panel of 4 genes were indeed up regulated by knockdown of DMBT1. ( E ) Four candidate genes (MMP-9, P-JUK, P-ERK, and P-AKT) were selected for western blot analysis. ( F ) luciferase activity of NF-κB pathway in DMBT1-transfected cells was increased, and decreased when overexpression of DMBT1.

Article Snippet: Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted with antibodies against Rabbit anti-human DMBT1 antibody and rabbit anti-humanC-IAP1 antibody (Cell Signaling Technology, CA, USA); antibodies of PI3K-AKT pathway which include: MMP-9, JUK, P-JUK, ERK, P-ERK, AKT, P-AKT(Cell Signaling Technology, CA, USA); normal mouse or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FLAG, mouse anti-G FP (Sigma, CA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Over Expression, Biomarker Discovery, RNA Sequencing, Luciferase, Activity Assay, Transfection

Journal: Oncotarget

Article Title: Long non-coding RNA CRNDE promotes gallbladder carcinoma carcinogenesis and as a scaffold of DMBT1 and C-IAP1 complexes to activating PI3K-AKT pathway

doi: 10.18632/oncotarget.12023

Figure Lengend Snippet: ( A – B ) RT-qPCR was used to detect the mRNA levels of CRNDE in 96 paired GBC tissues. CRNDE significantly upregulated in tumor tissues (β-actin used as an internal control). ( C – D ) RT-qPCR was used to detect the mRNA levels of c-IAP1 in 96 paired GBC tissues. c-IAP1 significantly upregulated in tumor tissues (β-actin used as an internal control). ( E ) Western blot and northern blot assay to detected the DMBT1, C-IAP1 and CRNDE expression level in five paired GBC tissues and NT tissues. ( F ) Correlation between DMBT1 and CRNDE in 42 paired GBC tissues. ( G ) Correlation between CRNDE and c-IAP1 in 42 paired GBC tissues.

Article Snippet: Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted with antibodies against Rabbit anti-human DMBT1 antibody and rabbit anti-humanC-IAP1 antibody (Cell Signaling Technology, CA, USA); antibodies of PI3K-AKT pathway which include: MMP-9, JUK, P-JUK, ERK, P-ERK, AKT, P-AKT(Cell Signaling Technology, CA, USA); normal mouse or rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FLAG, mouse anti-G FP (Sigma, CA, USA).

Techniques: Quantitative RT-PCR, Control, Western Blot, Northern Blot, Expressing