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: Cells

Article Title: p53 Promotes Cytokine Expression in Melanoma to Regulate Drug Resistance and Migration

doi: 10.3390/cells11030405

Figure Lengend Snippet: Reduced cytokine secretion in p53 knockout melanoma lines. ( A ) Comparative analysis of ligands abundance in supernatant from p53 ko and p53 WT LOX-IMVI cells by multiplex cytokine array. Differential expression of secreted ligand for each knockout clone compared to wild type is shown in the heatmap (as in log2-fold change). The observed ligand concentration (pg/mL) in p53 WT cells are depicted on the right side by numbers >10, 1–10, and <1. Cytokines analyzed by qPCR (in E and E) are indicated by an asterisk on the left side. ( B ) Expression of a subset of the ligands in wild-type and knockout clones by multiplex analysis is demonstrated. n = 3 (mean ± S.D), except for LIF, in which two biological samples had shown the value below the detection limit. ( C ) Validation of CXCL1 expression in p53 ko LOX-IMVI and SK-MEL-5 cells by Elisa analysis; n = 3 (mean ± S.D). Significance was determined by t -test in comparison to the wild-type group, and the observed level of significance is shown by asterisk (* < 0.05; ** < 0.005; *** < 0.0005; **** < 0.0001).

Article Snippet: Human CXCL1 expression was analyzed using Elisa set (R & D system, DY 275), according to company instruction.

Techniques: Knock-Out, Multiplex Assay, Quantitative Proteomics, Concentration Assay, Expressing, Clone Assay, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature Communications

Article Title: Nlrp12 mutation causes C57BL/6J strain-specific defect in neutrophil recruitment

doi: 10.1038/ncomms13180

Figure Lengend Snippet: ( a ) WT (B6N) or Nlrp12 −/− mice were infected i.n. with 5 × 10 3 colony-forming unit of F. tularensis LVS. Three days post infection BAL were performed and cytokine levels were determined by ELISA ( n =8, WT; n =9, Nlrp12 −/−) . ( b ) BMDM from WT (B6N) and Nlrp12 −/− mice were challenged with either F. tularensis LVS, S. aureus or P. aeruginosa . Eight hours later supernatants were collected and secretion of the indicated cytokine or chemokine quantified by ELISA. ( c ) BMDM from WT (B6N) and Nlrp12 −/− mice were stimulated with LPS (50 ng ml −1 ) for the indicated amount of time; supernatants were collected and assayed for CXCL1 production by ELISA. ( d ) BMDM from WT (B6N) and Nlrp12 −/− mice were left unstimulated or stimulated for 4 h with LPS in the presence of brefeldin A and monensin; intracellular CXCL1 was then assessed by flow cytometry. ( e ) BMDM from WT (B6N) and Nlrp12 −/− mice were stimulated with LPS for the indicated amount of time; Cxcl1 expression was quantified by real-time PCR. ( f ) BMDM from WT (B6N), WT (B6J) and Nlrp12 −/− mice were stimulated with LPS for 8 h; supernatants were collected and assayed for the indicated cytokines by ELISA. ( g ) BMDM from B6N, B6J, B6N-B6J F1 and B6N-B6J F2 mice were challenged with LPS for 8 h; supernatants were collected and CXCL1 secretion assessed by ELISA. The Nlrp12 allele was sequenced for all B6N-B6J F2 mice and cohorts stratified based on their Nlrp12 genotype ( Nlrp12 B6N/B6N , Nlrp12 B6N/B6J or Nlrp12 B6J/B6J ) ( n =14, B6N; n =13, B6J; n =14, F1; n =52, F2). Pooled data from three ( b , c ) independent experiments are depicted or are representative of two ( d ) or three ( e , f ) independent experiments. ( b , c , e , f ) Data are expressed as the mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.005, NS, not significant by Mann–Whitney U -test ( a , g ), Student's t -test ( b , e , f ) or two-way analysis of variance analysis ( c ).

Article Snippet: Antibody pairs for the IL-1β (MAB401 and BAF401; clone 30311 and polyclonal; R&D Systems; 8 and 4 μg ml −1 , respectively) and CXCL1 (mouse: MAB453 and BAF453; clone 48415 and polyclonal; R&D Systems; 8 and 0.4 μg ml −1 , respectively; human: MAB275, clone 20326, 4 μg ml −1 ; BAF275, polyclonal, 40 ng ml −1 ) and CCL3/ MIP-1α (Quantikine ELISA kit, Product #MMA00) ELISAs were from R&D Systems.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY