Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Immunogenic chemotherapy with cyclophosphamide and doxorubicin against established murine carcinoma

doi: 10.1007/s00262-009-0797-1

Figure Lengend Snippet: Partial role of calreticulin in anti-tumor effects induced by combination therapy with CP and DR. a BALB/c mice were injected s.c. with 2 × 105 CT-26 cells into the right flank and simultaneously vaccinated s.c. with DR-treated or MMC-treated 1 × 106 CT-26 cells into the left flank. Each group consisted of seven mice. *P < 0.05 indicates statistical significance. b CT-26 cells were transfected with calreticulin or control siRNA. Six days after transfection, the CT-26 cells were harvested, and immunoblot was performed. c As a protective model, BALB/c mice were injected s.c. in the left flank with DR-treated 1 × 106 CT-26 cells that were transfected with calreticulin or control siRNA. On day 14, mice were inoculated s.c. with 2 × 105 CT-26 cells into the right flank. Each group consisted of seven mice. The number in parentheses denotes tumor-rejected mice/total mice

Article Snippet: After centrifugation, proteins in the supernatant were loaded onto SDS-PAGE NuPAGE 4–12% Bis-Tris gels, transferred to the PVDF membrane using the iBlot dry blotting system (Invitrogen), and incubated with 1× EzBlock buffer (ATTO, Tokyo, Japan) in TBS-Tween for 60 min. After incubation with either anti-calreticulin (StressMarq Bioscience, Victoria, BC, Canada) or anti-β-actin (BioLegend) antibodies, membranes were incubated with the appropriate alkaline phosphatase-conjugated secondary antibodies and were developed by chemiluminescence (Invitrogen).

Techniques: Injection, Transfection, Western Blot

Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: HPV E7 interacts with IFI16 and the E3 ligaseTRIM21. (A) Mass spectrum data of TRIM21 among HPV 11E7-interacting proteins identified by mass spectrometry. (B) Immunoblot analysis of HaCaT cells stably expressing HPV 11E7 transfected with poly(dA:dT) for the indicated times, followed by immunoprecipitation with anti-FlagM2 beads. (C) Immunoblot analysis of HeLa cells transfected with poly(dA:dT) for the indicated times, followed by immunoprecipitation with IFI16, HPV 18E7, TRIM21 or immunoglobulin G (IgG)-conjugated magnetic beads. (D) Coimmunoprecipitation and immunoblot analysis of 293T cells cotransfected for 36 hr with Flag-IFI16 plus Myc-TRIM21 or Myc-TRIM21 mutant vectors, followed by immunoprecipitation with anti-Flag-M2 beads. (E) Coimmunoprecipitation and immunoblot analysis of 293T cells cotransfected for 36 hr with Flag-HPV 18E7 plus Myc-TRIM21 or Myc-TRIM21 mutant vectors, followed by immunoprecipitation with anti-FlagM2 beads. (F) Coimmunoprecipitation and immunoblot analysis of 293T cells cotransfected for 36 hr with Myc-TRIM21 and Flag-IFI16 or Flag-IFI16 mutant vectors, followed by immunoprecipitation with anti-FlagM2 beads.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Mass Spectrometry, Western Blot, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Magnetic Beads, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: HPV E7 promotes the K33-linked ubiquitination and degradation of IFI16 mediated by TRIM21. (A) Immunoblot analysis of lysates in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times. (B) Immunoblot analysis of IFI16 in the lysates of TRIM21 knockout stable HeLa cells treated with CHX (40 μg/ml) for the indicated number of hours after transfection with poly(dA:dT) for 1 hr. (C) Immunoblot analysis of 293T cells cotransfected with the Myc-TRIM21 and Flag-IFI16 vectors for 36 hr. (D) Immunoblot analysis of 293T cells cotransfected with the Myc-TRIM21 and Flag-IFI16 vectors with or without MG132 treatment. (E) Immunoblot analysis of 293T cells cotransfected with the Myc-TRIM21 and Flag-IFI16 vectors treated with CHX (40 μg/ml) for the indicated number of hours. (F) Immunoblot analysis of 293T cells cotransfected with Myc-TRIM21and the Flag-IFI16 or GFP-HPV 18E7 vector for 36 hr. (G) Immunoblot analysis of ubiquitinated IFI16 in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times and treated with MG132 for 6 hr before cell harvest. (H) Immunoblot analysis of ubiquitinated IFI16 in 293T cells cotransfected with Myc-TRIM21and the Flag-IFI16 or HA-ub vector for 36 hr with or without MG132 treatment for 6 hr before cell harvest. (I) Immunoblot analysis of ubiquitinated IFI16 in 293T cells transfected with combinations of the Myc-TRIM21, Flag-IFI16, HA-ub, and GFP-HPV 18E7 vectors for 36 hr and treated with MG132 for 6 hr before cell harvest. (J) Immunoblot analysis of ubiquitinated IFI16 in 293T cells transfected with combinations of the Myc-TRIM21, Flag-IFI16, HA-ub, and HA-ub mutant (each of the Lys residues were replaced by an Arg residue) vectors for 36 hr and treated with MG132 for 6 hr before cell harvest. (K) Immunoblot analysis of ubiquitinated IFI16 in 293T cells transfected with combinations of the Myc-TRIM21, Flag-IFI16, HA-ub, HA-ub mutant (all Lys residues but one was replaced with Arg residues) vectors for 36 hr and treated with MG132 for 6 hr before cell harvest. Data are representative of at least three independent experiments.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Knock-Out, Transfection, Plasmid Preparation, Mutagenesis, Residue

Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: TRIM21 downregulates cell pyroptosis induced by poly(dA:dT). (A) Microscopy imaging of cell death in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for 18 hr. (B) Flow cytometry analysis of propidium iodide-positive control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for 16 hr. (C) Cell viability assay in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times. (D) LDH assay in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times. (E) Immunoblot analysis of GSDMD and caspase-1 in the lysates of control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for 24 hr. (F) ELISA analysis of IL-18 and IL-1β in control HeLa cells or knockout stable HeLa cells transfected with poly(dA:dT) for 24 hr. Data are presented as mean ± SD of duplicate samples and are representative of at least three independent experiments. P values are determined by two-tailed Student's t test. ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Microscopy, Imaging, Control, Knock-Out, Transfection, Flow Cytometry, Positive Control, Viability Assay, Lactate Dehydrogenase Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: Schematic diagram showing that HPV E7 interacted with IFI16 and promoted the ubiquitin-mediated degradation of IFI16 by recruiting the E3 ligase TRIM21, resulting in the inhibition of cell pyroptosis during HPV infection.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Ubiquitin Proteomics, Inhibition, Infection

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Evolutionary conservation and sequence homology analysis of calreticulin across multiple species. ( A ) Phylogenetic analysis of calreticulin amino acid sequences from different species. The neighbor-joining phylogenetic tree was constructed using the bootstrap method in MEGA version 10.2.2 with 1000 bootstrap replicates. ( B ) Sequence homology analysis of calreticulin amino acid sequences among various species. The matrix shown represents a bidirectional pairwise comparison of calreticulin sequences. The upper triangular region displays the percentage of sequence identity (% identity), while the lower triangular region shows the corresponding percentage of sequence divergence (% divergence) between each pair of species. Black squares along the diagonal indicate self-alignments, where sequence identity is 100% and divergence is 0%.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Sequencing, Construct, Comparison

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Distribution of calreticulin in different goat organs. ( A ) Immunohistochemical staining illustrating the localization of calreticulin in the nasal mucosa, pharynx, trachea, lung, heart, liver, spleen, and small intestine of goats. Bars = 50 μm. ( B ) Analysis of calreticulin protein expression levels via Western blot in the nasal mucosa, pharynx, trachea, lung, heart, liver, spleen, and small intestine of goats. ( C ) The band obtained by Western blot was analyzed by gray value, and the result was expressed as the gray value ratios of the CALR/GAPDH. All data shown are the mean ± SD from three independent experiments. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Expression and purification of recombinant goat calreticulin using the Pichia pastoris expression system. ( A ) Schematic representation of the recombinant plasmid PpIC9K-CALR. ( B ) Agarose gel electrophoresis was performed to analyze the original recombinant plasmid (lane 1), the plasmid digested with EcoRI alone (lane 2), and the plasmid digested with both EcoRI and NotI (lane 3). ( C ) SDS-PAGE analysis of recombinant calreticulin expression in methanol-induced positive transformants, stained with Coomassie Brilliant Blue: Lane 1—culture supernatant of methanol-induced transformants; Lane 2—flow-through during Ni-NTA affinity purification; Lane 3—wash fraction; Lane 4—eluted protein with 200 mM imidazole; Lane 5—desalted and concentrated protein following ultrafiltration. ( D ) Western blot analysis confirming the identity of purified recombinant goat calreticulin (lane order as in panel ( C )). ( E ) SEC-HPLC analysis of the molecular weight and purity of yeast-secreted recombinant goat calreticulin. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Expressing, Purification, Recombinant, Plasmid Preparation, Agarose Gel Electrophoresis, SDS Page, Staining, Affinity Purification, Western Blot, Molecular Weight

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Antibacterial activity of recombinant goat calreticulin. ( A – C ) CFU assays were performed to evaluate the inhibitory effects of calreticulin against Escherichia coli , Salmonella typhimurium , and Pasteurella multocida . ( D – F ) Growth curve analysis of Escherichia coli , Salmonella typhimurium , and Pasteurella multocida in the presence of calreticulin to assess its impact on bacterial proliferation. All data are presented as mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA. ns, no significance; *** p < 0.001.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Activity Assay, Recombinant

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Recombinant goat calreticulin binds to and agglutinates bacteria. ( A – C ) ELISA analysis of bacterial binding: Escherichia coli , Salmonella typhimurium , and Pasteurella multocida (1 × 10 7 CFU/mL) were immobilized on microtiter plates and incubated with either CaCl 2 (10 mM), His-tag peptide (100 μg/mL), His-tag peptide (100 μg/mL) + CaCl 2 (10 mM), calreticulin (100 μg/mL), or calreticulin (100 μg/mL) + CaCl 2 (10 mM). Binding of calreticulin was detected using anti-His tag antibodies. ( D ) Western blot analysis of bacterial pellets after incubation with calreticulin (100 μg/mL) to confirm binding to Escherichia coli , Salmonella typhimurium , and Pasteurella multocida . ( E ) ELISA analysis of calreticulin binding activity to LPS. ( F ) Log-phase Escherichia coli , Salmonella typhimurium , and Pasteurella multocida were labeled with CFSE and incubated with calreticulin (100 μg/mL) ± 10 mM Ca 2+ at 37 °C for 2 h. Bacterial agglutination was visualized by fluorescence microscopy. Bars = 100 μm. All data are expressed as the mean ± SD from three independent experiments. Statistical significance was assessed using one-way ANOVA. ns, no significance; *** p < 0.001. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Recombinant, Bacteria, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Western Blot, Activity Assay, Labeling, Agglutination, Fluorescence, Microscopy

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Intranasal infection with Pasteurella multocida upregulates calreticulin expression in the respiratory tissues of the lambs. Thirty-day-old lambs were intranasally challenged with Pasteurella multocida and euthanized 24 h post-infection, after which respiratory tract tissues, including the nasal cavity, trachea, and lung, were collected for further analysis. ( A ) Immunohistochemical staining showing calreticulin expression in the nasal mucosa, trachea, and lungs following infection. Bars = 20 μm. ( B ) Quantitative analysis of immunohistochemical staining based on gray value measurements. ( C ) RT-qPCR analysis of calreticulin mRNA expression in different respiratory tissues after Pasteurella multocida infection. ( D ) Western blot analysis of calreticulin protein levels in infected respiratory tissues. ( E ) Densitometric analysis of Western blot results. All data are presented as mean ± SD from three independent experiments. Statistical significance was evaluated using one-way ANOVA. ** p < 0.01; *** p < 0.001. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Infection, Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Intranasal administration of calreticulin alleviates Pasteurella multocida -induced pathological injury and promotes bacterial clearance in the lambs. Thirty-day-old lambs were intranasally infected with Pasteurella multocida ; 6 h post-infection, 1 mL of recombinant calreticulin (2.5 mg/mL) was administered intranasally. Lambs were euthanized and necropsied at 24 h post-infection for sample collection. ( A ) Histopathological evaluation of nasal mucosa ( a – c ), trachea ( d – f ), and lung tissues ( g – i ) using H&E staining. Bars = 20 μm. ( B ) Average injury scores calculated for all lambs ( n = 3 replicates/group). ( C ) In situ hybridization with a Pasteurella multocida -specific fluorescent probe to detect bacterial load in nasal cavity ( a , b ), tracheal ( c , d ), and lung ( e , f ) tissue sections. Bars = 100 μm. ( D ) Quantification of fluorescence intensity derived from in situ hybridization results. All data are presented as mean ± SD from three independent experiments. Statistical significance was assessed using one-way ANOVA. *** p < 0.001.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Infection, Recombinant, Staining, In Situ Hybridization, Fluorescence, Derivative Assay

Journal: Scientific Reports

Article Title: Hypoxia activates Wnt/β-catenin signaling by regulating the expression of BCL9 in human hepatocellular carcinoma

doi: 10.1038/srep40446

Figure Lengend Snippet: Human HCC cell lines HepG2 and SMMC-7721 cells were cultured under the hypoxic condition for the indicated time periods. (A) The mRNA expression levels of BCL9 in these cells were determined by Taqman real-time PCR and normalized with actin. (B) The mRNA expression levels of VEGF in these cells were determined as a positive control. (C) The BCL9, HIF-1α and HIF-2α protein levels were determined by Western-blot assays. (D) Significant increase in TOPflash activity was observed under hypoxia treatment. Further, the TOPflash activity was significantly increased when BCL9 overexpressed while decreased when BCL9 knocked down. Data are presented as mean ± SD (n = 3).* p < 0.01, Student’s t -test.

Article Snippet: The antibody against HIF-1α (sc-13515, Santa Cruz Biotechnology, 1:500), antibody against HIF-2α (sc-13596, Santa Cruz Biotechnology, 1:500), antibody against BCL9 (ab37305, Abcam, 1:500 dilution) and antibody against β-actin (A5441, Sigma) were used in this study.

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Positive Control, Western Blot, Activity Assay

Journal: Scientific Reports

Article Title: Hypoxia activates Wnt/β-catenin signaling by regulating the expression of BCL9 in human hepatocellular carcinoma

doi: 10.1038/srep40446

Figure Lengend Snippet: ( A ) The human BCL9 gene contains 3 putative HREs in its promoter region. ( B ) Hypoxia activates the luciferase activity of reporter vectors containing HRE-B or HRE-C sites in the BCL9 promoter. HepG2 and SMMC-7721 cells were transfected with the luciferase reporter vectors, and then subjected to hypoxia treatment for 36 h before measuring luciferase activities. Luciferase reporter vectors containing the HRE site in the VEGF promoter was included as a positive control. ( C,D ) HIF-1α but not HIF-2α binds to HRE-B and HRE-C sites in the BCL9 promoter under the hypoxic condition in HepG2 cells as determined by ChIP assays. Cells were cultured under the hypoxic or normoxic conditions for 36 h before assays. The HRE site in the VEGF promoter serves as a positive control. The amount of DNA fragments pulled- down was determined by real-time PCR ( C ) or conventional PCR ( D ). ( E ) Ectopic HIF-1α expression increases BCL9 protein levels in HepG2 cells as determined by Western-blot assays. ( F,G ) HIF-1α binds to HRE-B and HRE-C sites in the BCL9 promoter in HepG2 cells transfected with HIF-1α expression plasmids as determined by ChIP assays. The amount of DNA fragments pulled-down was determined by real-time PCR ( F ) or conventional PCR ( F ). The HRE site in the VEGF promoter serves as a positive control. Data are presented as mean ± SD (n = 3). * p < 0.01 (Student’s t -test).

Article Snippet: The antibody against HIF-1α (sc-13515, Santa Cruz Biotechnology, 1:500), antibody against HIF-2α (sc-13596, Santa Cruz Biotechnology, 1:500), antibody against BCL9 (ab37305, Abcam, 1:500 dilution) and antibody against β-actin (A5441, Sigma) were used in this study.

Techniques: Luciferase, Activity Assay, Transfection, Positive Control, Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Hypoxia activates Wnt/β-catenin signaling by regulating the expression of BCL9 in human hepatocellular carcinoma

doi: 10.1038/srep40446

Figure Lengend Snippet: ( A ) Schematic overview depicting the targeting strategy for BCL9 promoter. Primers are shown as red boxes; the blue arrow indicates the cut site by the TALENs. Donor plasmids: CMV promoter, human cytomegalovirus (CMV) immediate early promoter gene; eGFP, enhanced green fluorescent protein gene; Below, scheme of BCL9 TALENs and their recognition sequence. TALE repeat domains are colored to indicate the identity of the repeat variable diresidue (RVD); each RVD is related to the cognate targeted DNA base by the following code (N1 = A, HD = C, NN = G, NG = T). (B) Genomic PCR and restriction digestion characterization of BCL9-p-ko HepG2 cells. (C) Different TALEN pairs were designed. (D) The activities of each two TALEN constructs were examined by SSA assay, and construct L1-R1 showed the highest activity among the constructs in the assay. (E) The genomic sequences around the target site of the clones were detected. (F) BCL9-wt, BCL9-p-ko and BCL9-ko HepG2 cells were cultured under the hypoxic condition. The BCL9 and HIF-1α protein levels were determined by Western-blot assays. Data are presented as mean ± SD (n = 3). * p < 0.01, Student’s t -test.

Article Snippet: The antibody against HIF-1α (sc-13515, Santa Cruz Biotechnology, 1:500), antibody against HIF-2α (sc-13596, Santa Cruz Biotechnology, 1:500), antibody against BCL9 (ab37305, Abcam, 1:500 dilution) and antibody against β-actin (A5441, Sigma) were used in this study.

Techniques: TALENs, Sequencing, Construct, SSA Assay, Activity Assay, Genomic Sequencing, Clone Assay, Cell Culture, Western Blot

Journal: Scientific Reports

Article Title: Hypoxia activates Wnt/β-catenin signaling by regulating the expression of BCL9 in human hepatocellular carcinoma

doi: 10.1038/srep40446

Figure Lengend Snippet: ( A ) The HepG2 cells infected with BCL9-wt, BCL9-p-ko and BCL9-ko were injected subcutaneously into the nude mice. (B) Mice were sacrificed to remove the tumors, and images were taken with a Nikon camera. (C) Tumor weight was evaluate among BCL9-wt, BCL9-p-ko and BCL9-ko group. (D) HIF-1α (red) and BCL9 (green) detected by FISH and IF in tumors: HIF-1α normally expressed in BCL9-wt, BCL9-p-ko and BCL9-ko groups; BCL9 normally expressed in BCL9-wt, weakly expressed in BCL9-p-ko group and unexpressed in BCL9-ko group. (E,F) Angiogenesis was detected by IHC and RT-PCR: angiogenesis was significantly decreased in BCL9-p-ko and BCL9-ko group. (G) WNT/β-catenin target genes, including CD44, Axin-2 and Survivin, were detected by RT-PCR. CD44, Axin-2 and Survivin were significantly decreased in BCL9-p-ko and BCL9-ko group. Data are presented as mean ± SD ( n = 3). * p < 0.01, Student’s t -test.

Article Snippet: The antibody against HIF-1α (sc-13515, Santa Cruz Biotechnology, 1:500), antibody against HIF-2α (sc-13596, Santa Cruz Biotechnology, 1:500), antibody against BCL9 (ab37305, Abcam, 1:500 dilution) and antibody against β-actin (A5441, Sigma) were used in this study.

Techniques: Infection, Injection, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Hypoxia activates Wnt/β-catenin signaling by regulating the expression of BCL9 in human hepatocellular carcinoma

doi: 10.1038/srep40446

Figure Lengend Snippet: HIF-1α and BCL9 protein levels were detected by IHC staining in tissue microarrays containing 488 cases of human HCC specimens. (A) HIF-1α (red) and BCL9 (green) detected by FISH and IF in human HCC specimens. (B) HIF-1α overexpression is associated with BCL9 overexpression in human HCC ( p < 0.000, Fisher exact test).

Article Snippet: The antibody against HIF-1α (sc-13515, Santa Cruz Biotechnology, 1:500), antibody against HIF-2α (sc-13596, Santa Cruz Biotechnology, 1:500), antibody against BCL9 (ab37305, Abcam, 1:500 dilution) and antibody against β-actin (A5441, Sigma) were used in this study.

Techniques: Immunohistochemistry, Over Expression