Journal: Molecular Cancer

Article Title: The homeoprotein DLX4 controls inducible nitric oxide synthase-mediated angiogenesis in ovarian cancer

doi: 10.1186/s12943-015-0368-3

Figure Lengend Snippet: DLX4 induces iNOS expression. (A) Staining of iNOS in sections of peritoneal tumors of mice that were inoculated with vector-control and +DLX4 ES2 lines. Bar, 20 μm. (B, C and D) Flow cytometric analysis of intracellular staining of DLX4 and iNOS in transfected ovarian cancer cell lines. Mean fluorescence intensities (MFI) of staining are indicated. Shown are representative examples of DLX4 and iNOS staining in (B) vector-control and +DLX4 ES2 cells, (C) vector-control and +DLX4 A2780 cells and (D) 2008 cells transfected with non-targeting shRNA and shRNAs that targeted two different regions of DLX4 (shDLX4-A, shDLX4-B). (E) qRT-PCR analysis of relative NOS2 mRNA levels in ES2 cells and NOS1, NOS2 and NOS3 mRNA levels in A2780 cells. Levels of each mRNA in +DLX4 cells are expressed relative to the level in vector-control cells. (F) qRT-PCR analysis of relative NOS1, NOS2 and NOS3 mRNA levels in 2008 cells. Levels of each mRNA in DLX4 shRNA-transfected cells are expressed relative to the level in non-targeting shRNA-transfected cells.

Article Snippet: Four-week-old female nude mice were purchased from Charles River and inoculated i.p. with 1 × 10 6 cells of ES2 lines (n = 5 mice per group).

Techniques: Expressing, Staining, Plasmid Preparation, Control, Transfection, Fluorescence, shRNA, Quantitative RT-PCR

Journal: Molecular Cancer

Article Title: The homeoprotein DLX4 controls inducible nitric oxide synthase-mediated angiogenesis in ovarian cancer

doi: 10.1186/s12943-015-0368-3

Figure Lengend Snippet: DLX4 stimulates STAT1 activity and induces iNOS expression in a STAT1-dependent manner. (A) qRT-PCR analysis of relative NOS2 mRNA levels in vector-control ES2 cells and in ES2 cells that expressed wild-type DLX4 or mutant DLX4 (DLX4-TA) with or without dominant-negative STAT1 (STAT1-dn). (B) Vector-control ES2 cells and ES2 cells that expressed wild-type or mutant DLX4 were transfected with a firefly luciferase reporter construct driven by GAS elements (GAS-LUC), stimulated without or with IFN-γ (10 ng/mL) for 16 h and then assayed for luciferase activity. (C) Activity of the GAS-LUC reporter construct was assayed in 2008 cells that expressed non-targeting and DLX4 shRNAs as described in (B) . (D) Western blot analysis of levels of total STAT1 and phosphorylated STAT1 in vector-control and +DLX4 ES2 cells that were stimulated with IFN-γ (10 ng/mL) for 0, 1, 6 and 18 h. (E) Lysates of U3A cell lines that lacked or stably expressed GFP-STAT1 fusion protein and/or FLAG-tagged DLX4 fused to GFP were assayed by Western blot using Abs to STAT1 and DLX4. (F) Activity of the GAS-LUC reporter construct in STAT1-deficient U3A cells and in U3A cells reconstituted with STAT1 that lacked or expressed DLX4. Transfected cells were stimulated with or without IFN-γ (10 ng/mL) for 16 h and then assayed for luciferase activity. (G) FLAG Ab was used to pull down FLAG-tagged DLX4 in U3A cells that were stimulated with IFN-γ (10 ng/mL) for 1 h. Immunoprecipitates were analyzed by Western blot using Ab to STAT1. Pulldown using control Ig was included as a negative control. Shown in B , C and F are relative firefly luciferase activities in three independent experiments.

Article Snippet: Four-week-old female nude mice were purchased from Charles River and inoculated i.p. with 1 × 10 6 cells of ES2 lines (n = 5 mice per group).

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation, Control, Mutagenesis, Dominant Negative Mutation, Transfection, Luciferase, Construct, Western Blot, Stable Transfection, Negative Control

Journal: Molecular Cancer

Article Title: The homeoprotein DLX4 controls inducible nitric oxide synthase-mediated angiogenesis in ovarian cancer

doi: 10.1186/s12943-015-0368-3

Figure Lengend Snippet: DLX4 stimulates NO levels, VEGF-A production and endothelial cell growth in vitro by inducing iNOS. (A) Flow cytometric analysis of iNOS levels in vector-control and +DLX4 ES2 cells, and in +DLX4 ES2 cells that stably expressed NOS2 shRNA (+DLX4 + shNOS2). (B) NO levels were assayed by using Griess reagent in culture medium that was conditioned by equivalent numbers of cells of each ES2 line. (C) Levels of VEGF-A were assayed by ELISA in tumor-conditioned medium. (D) Growth of endothelial cells cultured in tumor-conditioned medium was evaluated by MTT assay. Shown in B , C and D are average results of three independent experiments.

Article Snippet: Four-week-old female nude mice were purchased from Charles River and inoculated i.p. with 1 × 10 6 cells of ES2 lines (n = 5 mice per group).

Techniques: In Vitro, Plasmid Preparation, Control, Stable Transfection, shRNA, Enzyme-linked Immunosorbent Assay, Cell Culture, MTT Assay

Journal: Molecular Cancer

Article Title: The homeoprotein DLX4 controls inducible nitric oxide synthase-mediated angiogenesis in ovarian cancer

doi: 10.1186/s12943-015-0368-3

Figure Lengend Snippet: DLX4 stimulates tumor angiogenesis and ascites formation in i.p. xenograft models of ovarian cancer by inducing iNOS. Female nude mice (n = 5 per group) were inoculated i.p. with equivalent numbers of cells (1 x 10 6 ) of vector-control, +DLX4 and +DLX4 + shNOS2 ES2 lines and sacrificed at 20 days thereafter. (A) Volume of ascites. (B) Average numbers of microvessels were calculated in omental tumors by scoring five random 100x microscopic fields of CD34-stained tissue sections of each mouse. (C) Representative examples of CD34 staining in omental tumors. Bar, 100 μm.

Article Snippet: Four-week-old female nude mice were purchased from Charles River and inoculated i.p. with 1 × 10 6 cells of ES2 lines (n = 5 mice per group).

Techniques: Plasmid Preparation, Control, Staining

Journal: The Journal of Advanced Prosthodontics

Article Title: Repair bond strength of resin composite to bilayer dental ceramics

doi: 10.4047/jap.2018.10.2.101

Figure Lengend Snippet: Materials used in this study

Article Snippet: Clearfil Majesty ES-2 (A2) , light-curing restorative composite resin containing nano-fillers , , 170032 , Kuraray Noritake Dental Inc., Sakazu, Kurashiki, Okayama, Japan.

Techniques: Blocking Assay