Journal: PLoS ONE

Article Title: Stromal Cells Positively and Negatively Modulate the Growth of Cancer Cells: Stimulation via the PGE2-TNFα-IL-6 Pathway and Inhibition via Secreted GAPDH-E-Cadherin Interaction

doi: 10.1371/journal.pone.0119415

Figure Lengend Snippet: ( A ) MKN-7 and MKN-74 cells were cultured alone (Mono) or co-cultured with Hs738 (Cocul) for 3 days in the presence of MEK inhibitor I or U0126. Hs738 cells were cultured with the inhibitors for 2 days and CM was prepared. Both gastric cancer lines were cultured in the CM for 3 days. Cell growth was determined by measuring GFP fluorescence intensity. The values are means ± s.d. (n = 3). Cell growth is expressed as a percentage of the value without test compounds in each culture condition. ( B ) Hs738 cell extracts were incubated with b-MEK inh-pretreated streptavidin resin in the presence or absence of MEK inhibitor I (M) or U0126 (U) and the bound proteins were analyzed by Western blotting. L, 1/50 of loaded cell extracts. ( C ) Hs738 cells were cultured with MEK inhibitor I for 2 days and the cell lysates were analyzed by Western blotting. ( D ) Hs738 cells were cultured with MEK inhibitor I for 2 days and the concentrations of IL-6 and CXCL1 in the CM were determined. The values are means ± s.d. (n = 3). ( E ) Hs738 cells were treated with siRNA specific for RPL-18A (siRPL) or negative control (siCont) for 2 days and then re-inoculated followed by further culture for 2 days. The cell lysates were analyzed by Western blotting and the amounts of IL-6 in the CM were determined. The values are means ± s.d. (n = 3).

Article Snippet: Anti-human IL-6 neutralizing antibody (MAB206), recombinant human IL-6 (206-IL), and recombinant human CXCL1 (275-CR/CF) were purchased from R&D Systems.

Techniques: Cell Culture, Fluorescence, Incubation, Western Blot, Negative Control

Journal: PLoS ONE

Article Title: Stromal Cells Positively and Negatively Modulate the Growth of Cancer Cells: Stimulation via the PGE2-TNFα-IL-6 Pathway and Inhibition via Secreted GAPDH-E-Cadherin Interaction

doi: 10.1371/journal.pone.0119415

Figure Lengend Snippet: ( A ) MKN-7 and MKN-74 cells were cultured for 15 min with or without 50 μg/mL anti-IL-6 neutralizing antibody in Hs738 CM prepared by 2 days of culture. The activation of STAT3 was analyzed by Western blotting. ( B ) MKN-7 and MKN-74 cells were cultured for 15 min with IL-6. The activation of STAT3 was analyzed by Western blotting. ( C ) MKN-7 and MKN-74 cells were cultured with IL-6 or CXCL1 for 3 days. Cell growth was determined by measuring GFP fluorescence intensity. The values are means ± s.d. (n = 3). ( D ) MKN-7 and MKN-74 cells were cultured alone (Mono) or co-cultured with Hs738 cells (Cocul) with the indicated antibodies for 3 days. Cell growth was determined by measuring GFP fluorescence intensity. The values are means ± s.d. (n = 3). Cell growth is expressed as a percentage of the value without antibodies in each culture condition.

Article Snippet: Anti-human IL-6 neutralizing antibody (MAB206), recombinant human IL-6 (206-IL), and recombinant human CXCL1 (275-CR/CF) were purchased from R&D Systems.

Techniques: Cell Culture, Activation Assay, Western Blot, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Pyroglutamate and O -Linked Glycan Determine Functional Production of Anti-IL17A and Anti-IL22 Peptide-Antibody Bispecific Genetic Fusions

doi: 10.1074/jbc.M112.417717

Figure Lengend Snippet: The peptide-antibody genetic fusion neutralizes the IL22 activity equivalent to parental anti-IL22 antibody while lacking anti-IL17A neutralizing activity. A, kinetic analysis with human IL22 using a Biacore 2000. B, IL22-stimulated release of GRO-α production in HT29 cells. C, table summary of kinetic analysis with human IL17A using a Biacore 2000. D, IL17A-stimulated release of GRO-α in BJ human foreskin fibroblast cells. Error bars, S.D.

Article Snippet: Bound GRO-α was detected with anti-human GRO-α biotin (catalog no. BAF275, R&D Systems) at 40 ng/ml, followed by streptavidin-HRP (catalog no. DY998, R&D Systems), followed by a 1:1 mixture of H 2 O 2 and tetramethylbenzidine substrate (catalog no. DY999, R&D Systems), followed by 2 n H 2 SO 4 .

Techniques: Activity Assay

Journal: Cancer discovery

Article Title: Adenosine A2A Receptor Blockade as an Immunotherapy for Treatment-Refractory Renal Cell Cancer

doi: 10.1158/2159-8290.CD-19-0980

Figure Lengend Snippet: A-B) Adenosine signature related chemokine concentrations exhibited dose a dependent increase (CXCL1, panel A) or decrease (CXCL10, panel B). C) Addition of ciforadenant (1 μm) neutralizes the induction of CXCL5 by NECA as determined by ELISA. D-G) Purified human PBMCs from healthy donors were co-cultured with indicated the concentrations of NECA and were stimulated with anti-human CD3 and CD28 antibodies. Cells were kept in culture for 2 days. Golgi block was added 4 hours prior to collecting cells for intracellular flow cytometry analysis. CD14+ monocytic cells exhibited elevated expression of adenosine signature (as determined by mean fluorescence intensity, MFI) related cytokines and chemokines including CXCL5 (D), CCl2 (E), IL-8 (F), and CXCL1 (G) as NECA concentration increased. Lymphocytes including CD8+ T cells and CD19+ B cells had minimal changes. Error bars represent SEM.

Article Snippet: Antibodies used in analysis include: anti-human CD8a (Clone RPA-T8, BioLegend Cat No. 301048), anti-human CD3 (Clone OKT3, BioLegend Cat No. 317322), anti-human CD4 (clone OKT4, BioLegend Cat No. 317436), anti-human CD56 (clone 5.1H11, BioLegend Cat No. 362546), anti-human CD205 (clone HD30, BioLegend Cat No. 342210) anti-human CD14 (clone 63D3, BioLegend Cat No. 367118), anti-human CD19 (clone SJ25C1, BioLegend Cat No. 363034), anti-human CXCL5 (clone J111B7, BioLegend Cat No. 524104), anti-human MCP-1 (clone 2H5, BioLegend Cat No. 505904), anti-human IL-8 (clone E8N1, BioLegend Cat No. 511408), anti-human CXCL1 (clone 20326, R&D Systems Cat No. IC275P).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Cell Culture, Blocking Assay, Flow Cytometry, Expressing, Fluorescence, Concentration Assay