Journal: The Journal of Cell Biology

Article Title: Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo

doi: 10.1083/jcb.200611026

Figure Lengend Snippet: The N-terminal domain cleavage of fibulin-5 is found in aged mouse skin, as well as in cell cultures. (A) Fine structure of elastic fibers in skin tissues observed by transmission electromicroscopy. Elastin was stained with tannic acid, and therefore appears as black amorphous material. The fine fibers surrounding elastin are microfibrils. Bar, 0.4 μm. (B) Skin tissues were harvested from wild-type or fibulin-5–deficient young (3-mo-old) and old (22-mo-old) mice. Proteins were extracted from skin tissues with 8 M urea and dialyzed against PBS. 10 μg each of these extracts were resolved by SDS-PAGE, and analyzed by Western blotting with anti–fibulin-5 antibody (BSYN2473). Two specific bands of 45 and 55 kD were detected in wild-type mice with anti–fibulin-5 antibody (lanes 1–3). The 55-kD band markedly decreased with age, whereas the 45-kD band markedly increased with age (lanes 4–6). (C) 293T cells were transiently transfected with an expression vector encoding fibulin-5 cDNA with a signal peptide and a FLAG tag at the N terminus. Conditioned medium was subjected to SDS-PAGE, followed by Western blotting analysis with either anti– fibulin-5 or anti-FLAG antibody. Two bands of 55 and 45 kD were detected with anti–fibulin-5 antibody, whereas only a 55-kD band was detected with anti-FLAG antibody. (D) Anti–fibulin-5 antibody (BSYN2473) was raised against a peptide corresponding to amino acids 76–98 (red mark). These findings suggest that fibulin-5 is cleaved at a more N-terminal position than the recognition site of anti–fibulin-5 antibody.

Article Snippet: Protein samples of the same amount were subjected to SDS-PAGE (Invitrogen), followed by Western blotting with anti–fibulin-5 antibody (1/400, BSYN2473).

Techniques: Transmission Assay, Staining, SDS Page, Western Blot, Transfection, Expressing, Plasmid Preparation, FLAG-tag

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: Representative (A) RT-PCR and (B) Q-RT-PCR analyses of fibulin-5 expression in six-NPC samples (T) compared to that in three-normal tissues (N). GAPDH was used as internal control. (C) Western blot analyses of fibulin-5 protein expression level in NPC samples (T) and normal tissues (N) ( p < 0.001) Total proteins were extracted from respective tissues and probed with antibody against fibulin-5. β-actin was used as a control.

Article Snippet: Antibodies included against fibulin-5 (Epitomics), FLJ10540 (generated by us) [ , ], DDK (Origene), phosphor-AKT and AKT (Cell Signaling Technology, Beverely, MA, USA), lamin A/C, and β-actin (monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied.

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

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: (A) The intensity of fibulin-5 expression in various tissues by immunohistochemical staining were evaluated in (a) normal tissue and tumor tissues of NPC patients with stage I (b), II (c), III (d), IV (e), lymph node-negative (f), and lymph node-positive (g) samples. Original magnification, 200×. (B) Survival of patients with high fibulin-5 expression (solid line) was significantly abbreviated in comparison to those with low expression (dashed line), with a statistically significant difference in survival ( p = 0.003) by log-rank test.

Article Snippet: Antibodies included against fibulin-5 (Epitomics), FLJ10540 (generated by us) [ , ], DDK (Origene), phosphor-AKT and AKT (Cell Signaling Technology, Beverely, MA, USA), lamin A/C, and β-actin (monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: (A) Immunoblot analysis of total protein from normal tissues, TW01, TW04, and Hone1 cells using monoclonal anti-fibulin-5 antibody. (B) Hone1, A-431 and U2-OS cells were grown on glass cover slips and cultured overnight. Indirect immunofluorescence of endogenous fibulin-5 protein in Hone1 cells was detected by using anti-fibulin-5 antibody. (Bottom panel) Hone1 cells were transfected with DDK-fused fibulin-5 by Plus/Lipofectamine reagents and cultured overnight. Indirect immunofluorescence of highly expressed fibulin-5 protein in Hone1 cells was detected with anti-DDK antibody. The cells were double-stained with DAPI to detect DNA. (C) (Upper panel) Western blot analysis for fibulin-5 of cytoplasm extracts (left) and nuclear extracts (right) of TW01 and Hone1 cells. β-actin and LaminA/C were used as control. (Bottom panel) Immunoprecipitation analysis for fibulin-5 of cell culture supernatants and intra-cellular total cell extracts of TW01 and Hone1 cells.

Article Snippet: Antibodies included against fibulin-5 (Epitomics), FLJ10540 (generated by us) [ , ], DDK (Origene), phosphor-AKT and AKT (Cell Signaling Technology, Beverely, MA, USA), lamin A/C, and β-actin (monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied.

Techniques: Western Blot, Cell Culture, Immunofluorescence, Transfection, Staining, Immunoprecipitation

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: (A) DDK-tagged fibulin-5 was stably transfected into Hone1 cells, 2 clones were chosen, and DDK-fibulin-5 expression was determined by western blotting with anti-DDK and anti-β-actin antibodies. (B) Cell viability of Hone1 stable cell lines was measured by MTT assay. The cells were cultured for 0-3 days followed by MTT assay (OD 570 ) to quantitate the cell growth. Data were normalized against the OD 570 value on day 0 of each treatment. The results represent the mean ± SD of 3 independent experiments. (C) Wound healing assays with cells expressing empty vector or fibulin-5-Hone1. Representative images captured with a 10× objective at the time of wounding or 24 hour after. All experiments were repeated at least three times. The percentage of wound closure corresponds to the distance between wound edges in at least three randomly chosen regions relative to the distance at time 0 hour for each cell. (D) Migration and invasion of vehicle-Hone1 and fibulin-5-Hone1 stable cells (200×). For the migration assays, cells (vehicle-Hone1, fibulin-5-Hone1 stable clones) were seeded into the top of a Transwell insert. After 24 hour, the cells on top were scraped, and the cells that had migrated to the bottom were fixed and stained with Giemsa. The relative-fold migration values for the clones were normalized against the vehicle control and are represented diagrammatically. For the invasion assays, cells were seeded after the addition of Matrigel. The relative-fold invasion values for the stable clones were normalized against the vehicle cells and are represented diagrammatically.

Article Snippet: Antibodies included against fibulin-5 (Epitomics), FLJ10540 (generated by us) [ , ], DDK (Origene), phosphor-AKT and AKT (Cell Signaling Technology, Beverely, MA, USA), lamin A/C, and β-actin (monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied.

Techniques: Stable Transfection, Transfection, Clone Assay, Expressing, Western Blot, MTT Assay, Cell Culture, Plasmid Preparation, Migration, Staining

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: (A) Hone 1 cells were treated with indicated concentrations of fibulin-5, and cell growth was analyzed on days 0-3 by MTT assay. Data were normalized against the OD 570 value on day 1 of each treatment. The results represent the mean ± SD of 3 independent experiments. (B) Migration and invasion of Hone1 cells (200×). For the migration assays, Hone1 cells stimulated with indicate concentrations of fiblin-5 protein were seeded into the top of a Transwell insert. After 24 hour, the cells on top were scraped, and the cells that had migrated to the bottom were fixed and stained with Giemsa. The relative-fold migration values for the clones were normalized against the DMSO and are represented diagrammatically.

Article Snippet: Antibodies included against fibulin-5 (Epitomics), FLJ10540 (generated by us) [ , ], DDK (Origene), phosphor-AKT and AKT (Cell Signaling Technology, Beverely, MA, USA), lamin A/C, and β-actin (monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied.

Techniques: MTT Assay, Migration, Staining, Clone Assay

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: (A) A negative control siRNA plus fibulin-5 siRNA was transfected into Hone1 cells for 24 hour. After transfection, western blotting was performed with anti-fibulin-5 and β-actin antibodies. The mRNA expression level of endogenous fibulin-5 was also measured by Q-RT-PCR. (B) Using the same panel, the sifibulin-5 transfectants and negative control were seeded into 96-well plates with 5.0% FBS. The cells were cultured for 0-3 days followed by MTT assay to quantitate cell growth. The data were normalized against the value on day 0 of each treatment. The growth curves of Hone1 cells are shown as the mean ± SD of 3 independent experiments. (C and D) The wound healing, migration, and invasion results of negative control-Hone1 and sifibulin-5-Hone1 stable cells are shown (200×). The relative-fold migration and invasion of sifibulin-5-Hone1 cells were normalized against the values for the negative control cells and are represented diagrammatically. The results represent the mean ± SD of 3 independent experiments. The shFibulin-5-Hone-1 invasion panel of Figure 6D is excluded from this article's CC-BY license. See the accompanying retraction notice for more information.

Article Snippet: Antibodies included against fibulin-5 (Epitomics), FLJ10540 (generated by us) [ , ], DDK (Origene), phosphor-AKT and AKT (Cell Signaling Technology, Beverely, MA, USA), lamin A/C, and β-actin (monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied.

Techniques: Negative Control, Transfection, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, MTT Assay, Migration

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: (A) Vehicle-Hone1, fibulin-5-Hone1, vehicle-TW01, and fibulin-5-TW01 transfected cells were serum-starved and treated with the indicated inhibitors, SB202190, PD98059, and LY294002 or solvent for 24 hour. The migration and invasion ratios of vehicle-Hone1 and fibulin-5-Hone1 transfected cells were determined as previously described. (B) The fibulin-5 expressing cells of Hone1 and TW01 were serum-starved for 24 hour and treated with or without LY294002 at the final concentration of 10 μM. The Total cell lysates of vehicle-Hone1, fibulin-5-Hone1, vehicle-TW01, and fibulin-5-TW01 transfected cells were immunoblotted for the unphosphorylated and phosphorylated forms of AKT. β-actin was used as the internal loading control. (C) A negative control and fibulin-5 siRNAs were transfected into Hone1 cells for 24 hour and western blotting was performed as in (B).

Article Snippet: Antibodies included against fibulin-5 (Epitomics), FLJ10540 (generated by us) [ , ], DDK (Origene), phosphor-AKT and AKT (Cell Signaling Technology, Beverely, MA, USA), lamin A/C, and β-actin (monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied.

Techniques: Transfection, Migration, Expressing, Concentration Assay, Negative Control, Western Blot

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: (A) The mRNA and protein expression levels of FLJ10540 were determined by Q-RT-PCR and western blotting in fibulin-5 transfectants. The result of mRNA was normalized against the expression level of GAPDH mRNA in each fibulin-5-stable clones. For protein analyses, the cell lysates (50 μg) of Hone1/fibulin-5 transfectants was subjected to immunoblot analysis with anti-FLJ10540 and β-actin antibodies. β-actin was used as a control. (B) Fibulin-5 siRNAs decreased the expression levels of FLJ10540 mRNA and protein in fibulin-5-NPC transfectants. The Q-RT-PCR and western blotting were performed as in (B). (C) The luciferase assays were done to detect promoter activities of FLJ10540 in cotransfected with in a dose-dependent manner of DDK-, DDK-fibulin-5-, negative control and sifibulin-5 in Hone1 cells. The luciferase activity in 1μg cell lysate was normalized to β-galactosidase activity. Data are representative of three independent experiments done in triplicates. The Western blotting of DDK-fibulin-5 in a dose-dependent manner was shown in the right side of the left panel. (D) ChIP analysis of endogenous FLJ10540 promoter in the presence and absence of fibulin-5 in Hone 1 cells. The protein-DNA complexes were immunoprecipitated with DDK and IgG antibodies, and FLJ10540 promoter element was detected by PCR. (E) Migration and invasion decreased in cells transfected with FLJ10540 siRNA in vehicle-Hone1 and fibulin-5-Hone1 transfectants. The relative-fold migration and invasion values for the stable clones were normalized against the vehicle cells and are represented diagrammatically. All data represent the mean ± SD of 3 independent experiments.

Article Snippet: Antibodies included against fibulin-5 (Epitomics), FLJ10540 (generated by us) [ , ], DDK (Origene), phosphor-AKT and AKT (Cell Signaling Technology, Beverely, MA, USA), lamin A/C, and β-actin (monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Clone Assay, Luciferase, Negative Control, Activity Assay, Immunoprecipitation, Migration, Transfection

Journal: PLoS ONE

Article Title: Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

doi: 10.1371/journal.pone.0084218

Figure Lengend Snippet: (A) Hone1-expressing FLJ10540 cells were serum-starved for 24 hour and treated with or without AKT inhibitor. The Total cell lysates of vehicle-Hone and FLJ10540-Hone1, transfected cells were immunoblotted for the unphosphorylated and phosphorylated forms of AKT. β-actin was used as the internal loading control. (B) A negative control and FLJ10540 siRNAs were transfected into Hone1 cells for 24 hour and western blotting was performed as in (B). (C) Vehicle-Hone1 and FLJ10540-Hone1 transfected cells were serum-starved and treated with the AKT inhibitor for 24 hour. The migration and invasion ratios of vehicle-Hone1 and fibulin-5-Hone1 transfected cells were determined as previously described.

Article Snippet: Antibodies included against fibulin-5 (Epitomics), FLJ10540 (generated by us) [ , ], DDK (Origene), phosphor-AKT and AKT (Cell Signaling Technology, Beverely, MA, USA), lamin A/C, and β-actin (monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied.

Techniques: Expressing, Transfection, Negative Control, Western Blot, Migration

Journal: The Journal of Biological Chemistry

Article Title: Proteomic mapping reveals dysregulated angiogenesis in the cerebral arteries of rats with early-onset hypertension

doi: 10.1016/j.jbc.2023.105221

Figure Lengend Snippet: List of significantly regulated proteins detected in cerebral arteries from hypertensive rats compared to normotensive control

Article Snippet: Tissue sections were blocked in blocking buffer (5% normal swine serum (Jackson ImmunoResearch), 1% bovine serum albumin (BSA, Sigma), 0.1% TritonX-100 (Sigma) in 1 × PBS) and stained with commercial anti-alpha smooth muscle actin antibody [1A4] (ab7817, 1:400, Abcam), anti-Fibulin 5 antibody (ab202977, 1:200, Abcam), and anti-human cadherin-13 antibody (AF3264, 1:200, R&D Systems) diluted in 1% BSA, 0.1% TritonX-100 in 1×PBS overnight at 4 °C.

Techniques: Molecular Weight, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Proteomic mapping reveals dysregulated angiogenesis in the cerebral arteries of rats with early-onset hypertension

doi: 10.1016/j.jbc.2023.105221

Figure Lengend Snippet: Differential expression of two candidate proteins across vascular beds. A , Venn diagram showing number of angiogenesis-associated proteins in cerebral arteries that are shared with mesenteric and renal arteries but change in opposite direction. B , column graph depicting logFC difference of Fbln5 (= blue ) and Cdh13 (= gray ) in cerebral, renal, and mesenteric artery comparisons between 12-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). C – G , immunohistochemistry analysis of α-SMA ( green ), Fbln5 ( red ), Cdh13 ( magenta ) in cerebral arteries from WKY controls and SHRs (n = 4 biological replicates, 2–4 technical replicates per rat). Scale bar represents 20 μm. Max fluorescent intensity was measured across a line. SEM is included on column graphs (WKY = blue , SHR = orange ).

Article Snippet: Tissue sections were blocked in blocking buffer (5% normal swine serum (Jackson ImmunoResearch), 1% bovine serum albumin (BSA, Sigma), 0.1% TritonX-100 (Sigma) in 1 × PBS) and stained with commercial anti-alpha smooth muscle actin antibody [1A4] (ab7817, 1:400, Abcam), anti-Fibulin 5 antibody (ab202977, 1:200, Abcam), and anti-human cadherin-13 antibody (AF3264, 1:200, R&D Systems) diluted in 1% BSA, 0.1% TritonX-100 in 1×PBS overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry