Journal:

Article Title: SHORT PEDF-DERIVED PEPTIDE INHIBITS ANGIOGENESIS AND TUMOR GROWTH

doi: 10.1158/1078-0432.CCR-08-2113

Figure Lengend Snippet: (A) Representative endothelial cell (HUVEC) chemotaxis assays: P14, P18 and P23 were tested at increasing concentrations alone (□) or in combination with bFGF (Δ). The assay was performed independently for each peptide; each peptide concentration was tested in quadruplicate to achieve statistical significance and the experiment repeated three times. Values that differ significantly from the background control (BSA alone, P<0.001) are indicated with asterisks. Note the inhibition of bFGF-induced chemotaxis by P14, P18 but not P23. hpf, high-powered fields. (B-D) The induction of endothelial cell (HMVEC) apoptosis by PEDF peptides: the cells were plated on coverslips and incubated overnight with P14, P18 and P23 in low-serum medium alone, or in the presence of protective bFGF. Apoptosis was detected by TUNEL, PEDF (10 nM) and the 34-mer (100 nM) were used as control. Representative images are shown (B). (C) Neither of the peptides significantly augmented apoptosis by serum withdrawal (P=0.06, top). However, P18 and P23 overcame the protective effect of bFGF (P<0.008, bottom, asterisks indicate P<0.05). Note the lack of apoptosis in the presence of P14. Values are shown as mean +/−SD. (D) P18 had no effect on tumor cells. PC-3 and Renca cells were treated with increasing concentrations of P18 and the numbers of viable cells evaluated by MTT assay. Note the lack of significant differences in cell numbers with increasing P18 concentration.

Article Snippet: After 48 hours the number of viable cells was assessed using MTT assay kit (Biotium Inc., Hayward CA) according to the manufacturer’s instructions.

Techniques: Chemotaxis Assay, Concentration Assay, Inhibition, Incubation, TUNEL Assay, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of IL-13 and IL-13Rα2 Expression by IL-32θ in Human Monocytic Cells Requires PKCδ and STAT3 Association

doi: 10.3390/ijms20081949

Figure Lengend Snippet: List of Interleukin (IL)-32θ down-regulated genes identified by microarray analysis.

Article Snippet: The IL-13 promoter (−940 to +48) luciferase vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Microarray, Chemotaxis Assay, Migration, Transduction, Coagulation, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of IL-13 and IL-13Rα2 Expression by IL-32θ in Human Monocytic Cells Requires PKCδ and STAT3 Association

doi: 10.3390/ijms20081949

Figure Lengend Snippet: IL-32θ inhibits IL-13 and IL-13Rα2 expression. ( A ) Constitutive expression of IL-32θ in stably expressing THP-1 cell model was confirmed by RT-PCR and Western blotting analyses. ( B ) mRNA expression of IL-13, IL-13Rα1, and IL-13Rα2 in THP-1/EV (empty vector) or THP-1/IL-32θ upon phorbol 12-myristate 13-acetate (PMA) (10 nM) or IL-13 (20 ng/ mL) stimulation after 24 h ( n = 4). ( C ) Secretion level of IL-13 after PMA treatment for 24 h was measured by ELISA ( n = 3). ( D ) Flow cytometry analysis was performed to assess the cell surface expression of IL-13Rα2 by using the anti-human IL-13Rα2 antibody. Data are shown as mean ± SEM. Statistical significance was analyzed using two-way ANOVA test followed by multiple comparison tests (* p < 0.05). Results are representative of single experiments.

Article Snippet: The IL-13 promoter (−940 to +48) luciferase vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of IL-13 and IL-13Rα2 Expression by IL-32θ in Human Monocytic Cells Requires PKCδ and STAT3 Association

doi: 10.3390/ijms20081949

Figure Lengend Snippet: Promoter analysis of IL-13 and IL-13Rα2 containing STAT3 and STAT6 binding sites. ( A ) Left panel, diagram showing putative element sites for STAT3 or STAT6 binding to promoters. Right panel, the luciferase assay was analyzed after transfecting IL-13 and IL-13Rα2 promoter reporter plasmids to THP-1/EV or THP-1/IL-32θ cells followed by 10 nM PMA treatment ( n = 4). ( B , C ) Chromatin immunoprecipitation (ChIP) assay was performed by using the DNA eluted from the immunoprecipitation of STAT3 and STAT6. Quantitative PCR results ( n = 3) were obtained by running primers designed to target three binding sites of STAT3 or STAT6 on the IL-13 promoter ( B ) and two binding sites on the IL-13Rα2 promoter ( C ). Data are shown as mean ± SEM. Statistical significance was analyzed using two-way ANOVA test followed by multiple comparison tests (* p < 0.05). Results are representative of single experiments.

Article Snippet: The IL-13 promoter (−940 to +48) luciferase vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Binding Assay, Luciferase, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of IL-13 and IL-13Rα2 Expression by IL-32θ in Human Monocytic Cells Requires PKCδ and STAT3 Association

doi: 10.3390/ijms20081949

Figure Lengend Snippet: Blocking STAT3 or STAT6 binding activities by decoy oligodeoxynucleotides (ODNs). THP-1/EV and THP-1/IL-32θ cells were transfected with STAT3 or STAT6 or scrambled decoy ODNs for 24h followed by treatment with PMA (10 nM) for 1 h before being prepared for further analyses. ( A ) Western blot result of the nuclear protein probed with anti-STAT3 or STAT6 antibodies ( n = 3). ( B ) mRNA expression of IL-13 and IL-13Rα2 conducted by RT-qPCR ( n = 3). ( C ) Relative luciferase activities of IL-13 and IL-13Rα2 promoters ( n = 4). Data are shown as mean ± SEM. Statistical significance was analyzed using two-way ANOVA test followed by multiple comparison tests (* p < 0.05). Western blot bands were quantified by Fiji software. Results are representative of single experiments.

Article Snippet: The IL-13 promoter (−940 to +48) luciferase vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Blocking Assay, Binding Assay, Transfection, Western Blot, Expressing, Quantitative RT-PCR, Luciferase, Software

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of IL-13 and IL-13Rα2 Expression by IL-32θ in Human Monocytic Cells Requires PKCδ and STAT3 Association

doi: 10.3390/ijms20081949

Figure Lengend Snippet: Signaling pathways for the IL-32θ-mediated down-regulation of IL-13 and IL-13Rα2 expression in THP-1 cells. Briefly, IL-13 stimulation causes the activation of both STAT3 and STAT6 to regulate IL-13 expression, but not IL-13Rα2, while PMA stimulation can activate both transcription factors and trigger IL-13 and IL-13Rα2 expression. IL-32θ suppresses IL-13 and IL-13Rα2 by interacting directly with PKCδ and STAT3 and subsequently reduces STAT3 binding activity to promoters. STAT6 activity may be partly and indirectly disturbed by IL-32θ. “Y” stands for Tyrosine. “S” stands for Serine.

Article Snippet: The IL-13 promoter (−940 to +48) luciferase vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Activation Assay, Binding Assay, Activity Assay