Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: (A) Representative results of CSL-siRNA inhibition on leptin upregulation of protein levels of Notch 1-3, activated NICD1 and Notch targeted genes, CSL, survivin and Hey2 in 4T1 cells as determined by western blot (WB). The WB results were normalized to β-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software for (B) CSL; (C) Notch1; (D) NICD1; (E) Notch 2; (F) Notch 3; (G) Hey2 and (H) survivin. Cells cotransfected with siRNA oligonucleotides (CSL-siRNA or control-siRNA) were treated with leptin (0 and 1.2 nM) for 24 h. (a) P<0.05 when comparing protein levels to cells treated with control-siRNA (basal) or CSL-siRNA. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: Polyclonal anti-Notch4, anti-OB-R-NH2 antibodies, monoclonal anti-IL-1R tI, nonspecific goat IgG2b, CSL small interference (si)RNA and control siRNA-A were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Inhibition, Western Blot, Software, Derivative Assay

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: Leptin transcriptional activation of CBF (or CSL) promoter is linked to several leptin-induced signaling pathways. (A) 4T1 cells were transiently transfected with a CBF-Luc reporter construct and incubated with leptin (0 and 1.2 nM) alone or plus siRNA oligonucleotides (SignalSilence control-siRNA and STAT3, MEK1, AKT1, mTOR, JNK, p38-siRNA). (B) Protein levels of kinases after siRNA treatment were determined by WB analysis using β-actin as loading control. (C) 4T1 cells transfected with CBF-Luc reporter were incubated with leptin alone or plus pharmacological inhibitors of JAK2/STAT3 (AG490, 30 µM), MEK/MAPK/ERK1/2 (PD98059, 30 µΜ), PI-3K/AKT1 (Wortmannin, 50 nM), PKC-Ca dependant (Gö6976, 30 µΜ), p38 kinase (SB203580, 2 µΜ), JNK (SP600125, 30 µM) and mTOR (Rapamycin, 20 nM) signaling pathways for 24 h. Luciferase activity was determined as described (see M &M) and expressed as a percent of basal or leptin-treated cells. (a) P<0.05 when comparing levels of luciferase activity to control (basal) or leptin-treated cells. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: Polyclonal anti-Notch4, anti-OB-R-NH2 antibodies, monoclonal anti-IL-1R tI, nonspecific goat IgG2b, CSL small interference (si)RNA and control siRNA-A were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Activation Assay, Transfection, Construct, Incubation, Luciferase, Activity Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: A) Representative results from immunocytochemistry (ICC, hematoxylin staining) of leptin and DAPT effects on migration of 4T1 cells as compared to basal conditions. B) Quantitative assessment of cell migration under the effects of leptin and DAPT. C) Representative results from ICC after addition of control-siRNA (Ctr-siRNA), CLS-siRNA and leptin. D) Quantitative assessment of cell migration under the effects of leptin, CSL-siRNA and Crt-siRNA. E) Effects of leptin and DAPT on 4T1 cell proliferation. F) Effects of leptin, Ctr-siRNA and CSL-siRNA on 4T1 cell proliferation. Results from cell migration (Boyden chamber cell migration assay) and proliferation (MTT cell proliferation assay) were obtained after 24 h and normalized to basal conditions (see Material and Methods). Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: Polyclonal anti-Notch4, anti-OB-R-NH2 antibodies, monoclonal anti-IL-1R tI, nonspecific goat IgG2b, CSL small interference (si)RNA and control siRNA-A were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Immunocytochemistry, Staining, Migration, Cell Migration Assay, MTT Cell Proliferation, Derivative Assay

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: (A) DAPT inhibition of γ-secretase abrogated leptin transcriptional induction of VEGF (A); VEGFR-2 (B) and IL-1α mRNA (C) as determined by real-time RT-PCR and normalized to the GAPDH expression. CSL-siRNA also inhibited leptin-induced effects on protein levels of VEGF (D); VEGFR-2 (E) and IL-1α (F) mRNA. Representative results of DAPT (G) and CSL-siRNA (M) inhibition of leptin upregulation of protein levels of VEGF, VEGFR-2, IL-1α, ERα and OB-R in 4T1 cells as determined by western blot (WB). The WB results were normalized to β-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software for VEGF (H and N); VEGFR-2 (I and O); IL-1α (J and P); ERα (K and Q) and OB-R (L and R) in cells treated with leptin (0 and 1.2 nM) and DAPT for 24 h or cotransfected with CSL-siRNA and control (Ctr)-SiRNA, respectively. (a) P<0.05 when comparing levels of antigens to basal conditions and control-siRNA. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: Polyclonal anti-Notch4, anti-OB-R-NH2 antibodies, monoclonal anti-IL-1R tI, nonspecific goat IgG2b, CSL small interference (si)RNA and control siRNA-A were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Western Blot, Software, Derivative Assay

Journal:

Article Title: Targeting Improves the Efficacy of a DNA Vaccine against Corynebacterium pseudotuberculosis in Sheep

doi:

Figure Lengend Snippet: Kinetics of production of antibody to hIg following immunization. Animals were bled weekly, and the sera were tested for the presence of antibodies to hIg by a specific ELISA. The groups of animals that had significantly (P < 0.05) higher titers of antibody to hIg compared to control groups are indicated by asterisks, and the relevant minimum (Min), maximum (Max), and median values are shown. W, week.

Article Snippet: For the detection of anti-hIg antibodies, the plates were coated with 5 μg of hIg protein (CSL Limited)/ml.

Techniques: Enzyme-linked Immunosorbent Assay