Journal: International journal of oncology

Article Title: Expression of TWEAK/Fn14 in neuroblastoma: implications in tumorigenesis.

doi: 10.3892/ijo.2013.1800

Figure Lengend Snippet: Figure 1. TWEAK and Fn14 expression and secretion in neuroblastoma cell lines and primary tumors. (A) RT-PCR showed expression of TWEAK and Fn14 in all neuroblastoma cell lines investigated. NTC, negative control. (B) Western blot analysis detected protein bands of ~30-35 kDa corresponding to TWEAK and 14-17 kDa corresponding to Fn14 in neuroblastoma cell lines. β-actin was used to ensure equal loading. (C) TWEAK secretion in neuroblastoma cells. SK-N-AS cells were treated with 10 or 50 ng/ml IL-1β and TNF-α and cell supernatants were analyzed for TWEAK secretion measured by ELISA. (D) Immunofluorescence images of neuroblastoma cell line SK-N-AS showing cellular distribution of TWEAK (red) and Fn14 (green). Merge shows coloca lization of ligand and receptor. DAPI shows staining of the nucleus. (E) Immunohistochemical detection of TWEAK (x400), Fn14 (x200) and phospho-NF-κB (x200) in primary human neuroblastoma tumor tissue (H/E hematoxylin/eosin; x400).

Article Snippet: Cells were transfected with target-specific Fn14 (sc-43764), TWEAK (sc-37522), control (Fluorescein conjugate-A) (sc-36869) or scrambled control (sc-37007) siRNA (Santa Cruz Biotechnology), respectively, at a concentration of 33 nM using Lipofectamine 2000 in OptiMEM.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Immunohistochemical staining

Journal: International journal of oncology

Article Title: Expression of TWEAK/Fn14 in neuroblastoma: implications in tumorigenesis.

doi: 10.3892/ijo.2013.1800

Figure Lengend Snippet: Figure 2. Fold scatter plot of TWEAK (TNFSF12) and Fn14 (TNFRSF12A) mRNA expression in primary tumors. The log2 fold between high stage (INSS stage 3-4) and low stage (INSS stage 1-2) tumors from four Affymetrix microarray studies, presented as three data sets are plotted. The log2 fold mean in each group is marked by horizontal line and related to the mean in low stage tumors (log2 fold, 0). Open circles, Versteeg data set (28) from the r2 database data set (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi. Open squares, McArdle/Wilzén data set (26,27). Open triangles, De Preter data set (25). N, the number of cases. FC, relative fold change between groups. p, significance by Welch's t-test (*p<0.05). See text for more detail.

Article Snippet: Cells were transfected with target-specific Fn14 (sc-43764), TWEAK (sc-37522), control (Fluorescein conjugate-A) (sc-36869) or scrambled control (sc-37007) siRNA (Santa Cruz Biotechnology), respectively, at a concentration of 33 nM using Lipofectamine 2000 in OptiMEM.

Techniques: Expressing, Microarray

Journal: International journal of oncology

Article Title: Expression of TWEAK/Fn14 in neuroblastoma: implications in tumorigenesis.

doi: 10.3892/ijo.2013.1800

Figure Lengend Snippet: Figure 3. Effect of TWEAK treatment on NF-κB cellular localization in neuroblastoma cells. (A) NF-κB immunostaining of TWEAK-stimulated neuroblastoma cells. SK-N-AS cells were cultured under reduced serum conditions (0.1%) for 16 h prior to stimulation with TWEAK (100 ng/ml). Cells were fixed and immunostained for the p65 subunit of NF-κB. The left panel shows non-treated SK-N-AS cells, whereas the right panel shows cells treated with TWEAK for 20 min. (B) TWEAK stimulates NF-κB p65 phosphorylation in neuroblastoma cells. SK-N-AS cells were cultured under reduced serum conditions (0.1%) for 16 h prior to stimulation with TWEAK (100 ng/ml) for various time periods as indicated or reduced serum for control. Following treatment cells were lysed and total cell extracts were immunoblotted for both the phospho-NF-κB and NF-κB subunit. β-actin was used as a control for equal loading. (C) TWEAK induces NF-κB transcrip tional activity in neuroblastoma cells. SK-N-AS cells transiently transfected with a NF-κB plasmid containing luciferase reporter gene were exposed to TWEAK (100 ng/ml), TNF-α (30 ng/ml) or culture medium only (control) for 6 h. Luciferase activity was measured in cell lysates and values were normalized to total protein content. The experiment was repeated three times with similar results. Data presented are mean ± SEM from a representative experiment, n=3 per group.

Article Snippet: Cells were transfected with target-specific Fn14 (sc-43764), TWEAK (sc-37522), control (Fluorescein conjugate-A) (sc-36869) or scrambled control (sc-37007) siRNA (Santa Cruz Biotechnology), respectively, at a concentration of 33 nM using Lipofectamine 2000 in OptiMEM.

Techniques: Immunostaining, Cell Culture, Phospho-proteomics, Control, Activity Assay, Transfection, Plasmid Preparation, Luciferase

Journal: International journal of oncology

Article Title: Expression of TWEAK/Fn14 in neuroblastoma: implications in tumorigenesis.

doi: 10.3892/ijo.2013.1800

Figure Lengend Snippet: Figure 4. Silencing of TWEAK and Fn14 in neuroblastoma cells. (A) TWEAK and Fn14 expression are important for neuroblastoma growth. Trypan blue exclusion assay performed 72 h after initial transfection showing cell viability upon transfection of SK-N-AS cells with siRNA towards TWEAK or Fn14 in addition to siRNA transfection followed by recombinant TWEAK treatment (p<0.05). (B) Expression of TWEAK and Fn14 in SK-N-AS cells after transfection with scramble siRNA and siRNA towards TWEAK or Fn14. (C) Cell viability assay results were verified by western blot analysis showing a decrease in the endogenous protein expression of TWEAK in SK-N-BE(2) cells and Fn14 in SK-N-AS cells upon silencing with siRNA.

Article Snippet: Cells were transfected with target-specific Fn14 (sc-43764), TWEAK (sc-37522), control (Fluorescein conjugate-A) (sc-36869) or scrambled control (sc-37007) siRNA (Santa Cruz Biotechnology), respectively, at a concentration of 33 nM using Lipofectamine 2000 in OptiMEM.

Techniques: Expressing, Trypan Blue Exclusion Assay, Transfection, Recombinant, Viability Assay, Western Blot

Journal:

Article Title: Mutations of the Membrane-Bound Disulfide Reductase DsbD That Block Electron Transfer Steps from Cytoplasm to Periplasm in Escherichia coli

doi: 10.1128/JB.00368-06

Figure Lengend Snippet: Sequence alignments of membrane-embedded domains of CcdA and DsbD. The strains having CcdA, DsbD containing COG4233, or DsbD are denoted with a superscript a, b, or c, respectively. The residue number of E. coli DsbD was calculated from the mature DsbD. Rho_cap represents Rhodobacter capsulatus; the accession number of CcdA is AAF26218, available from the ERGO-Light Database of Integrated Genomics (http://www.ergo-light.com/). Str_coe represents Streptomyces coelicolor A3(2); the DDBJ/EMBL/GenBank accession number is NP_628639. Oce_ihe represents Oceanobacillus iheyensis HTE831, accession number NP_692598; Met_maz represents Methanosarcina mazei Goe1, accession number NP_634265; Glo_vio represents Gloeobacter violaceus, accession number NP_926909; Cau_cre represents Caulobacter crescentus CB15, accession number NP_419036; Chl_pne represents Chlamydophila pneumoniae TW-183, accession number NP_877086; Sal_ent represents Salmonella enterica subsp. enterica serovar Typhimurium, accession number NP_455609; Esc_col represents Escherichia coli K12, accession number NP_418559; Nit_eur represents Nitrosomonas europaea, accession numbers ATCC 19718 and NP_842384; Pse_aer represents Pseudomonas aeruginosa PAO1, accession number AAG05866; and Cam_jej represents Campylobacter jejuni subsp. jejuni, accession numbers NCTC 11168 and NP_281786.

Article Snippet: Oce_ihe represents Oceanobacillus iheyensis HTE831, accession number {"type":"entrez-protein","attrs":{"text":"NP_692598","term_id":"23099132","term_text":"NP_692598"}} NP_692598 ; Met_maz represents Methanosarcina mazei Goe1, accession number {"type":"entrez-protein","attrs":{"text":"NP_634265","term_id":"21228343","term_text":"NP_634265"}} NP_634265 ; Glo_vio represents Gloeobacter violaceus , accession number {"type":"entrez-protein","attrs":{"text":"NP_926909","term_id":"37523532","term_text":"NP_926909"}} NP_926909 ; Cau_cre represents Caulobacter crescentus CB15, accession number {"type":"entrez-protein","attrs":{"text":"NP_419036","term_id":"16124472","term_text":"NP_419036"}} NP_419036 ; Chl_pne represents Chlamydophila pneumoniae TW-183, accession number {"type":"entrez-protein","attrs":{"text":"NP_877086","term_id":"33242145","term_text":"NP_877086"}} NP_877086 ; Sal_ent represents Salmonella enterica subsp. enterica serovar Typhimurium, accession number {"type":"entrez-protein","attrs":{"text":"NP_455609","term_id":"16759992","term_text":"NP_455609"}} NP_455609 ; Esc_col represents Escherichia coli K12, accession number {"type":"entrez-protein","attrs":{"text":"NP_418559","term_id":"16131961","term_text":"NP_418559"}} NP_418559 ; Nit_eur represents Nitrosomonas europaea , accession numbers ATCC 19718 and {"type":"entrez-protein","attrs":{"text":"NP_842384","term_id":"30250314","term_text":"NP_842384"}} NP_842384 ; Pse_aer represents Pseudomonas aeruginosa PAO1, accession number {"type":"entrez-protein","attrs":{"text":"AAG05866","term_id":"9948529","term_text":"AAG05866"}} AAG05866 ; and Cam_jej represents Campylobacter jejuni subsp. jejuni , accession numbers NCTC 11168 and {"type":"entrez-protein","attrs":{"text":"NP_281786","term_id":"15791963","term_text":"NP_281786"}} NP_281786 .

Techniques: Sequencing