Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 2. Activity of selected aptamer clones in a HEPG2 re- porter gene cell line. OSM signaling via the OSM receptor in the HEPG2 reporter cell line results in production of secreted alkaline phosphatase. Aptamer clones ADR58 (L), 120 (E), and 152 (M) have affinities of ,10 nM for OSM; ADR147 (f) is a negative control, and ADR66 (Œ) is an aptamer with an affinity .1000 nM for OSM. Aptamers were preincubated with 2 ng/ml OSM (which is approximately the ED50 for this assay), and production of secreted alkaline phosphatase was monitored following addition of OSM/aptamer mixture to the HEPG2 reporter cell line. The experiment was carried out in triplicate. Error bars represent S.D.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Activity Assay, Clone Assay, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 6. Effect of aptamers on LIF- and OSM-induced interleu- kin 6 release from KB cells. Shown are ADR58 (f) and ADR147 control (Œ). Aptamers were preincubated with either 1 ng/ml LIF (a) or 1 ng/ml OSM (b). The mixture was incubated with KB cells for 24 h. Interleukin 6 levels in the cell culture medium were then measured by ELISA. The experiment was carried out in triplicate. Error bars repre- sent S.D. The concentrations of LIF and OSM used were at the approx- imate ED50 for this assay. In the absence of aptamer, the IL-6 release induced by 1 ng/ml LIF was 1113 6 7.5 pg/ml, and the release induced by OSM was 4702 6 67 pg/ml. From cells incubated with medium alone, the IL-6 release was 49 6 9 pg/ml.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Control, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 7. Effect of aptamer on IL-6 signaling in the HEPG2 re- porter gene cell line. IL-6 signaling via the IL-6 receptor in the HEPG2 reporter cell line results in production of secreted alkaline phosphatase. Shown are ADR58 (f) and ADR147 control (Œ). Aptamer and control RNA were preincubated with 4 ng/ml IL-6 (which is approx- imately the ED50 for this assay), and production of secreted alkaline phosphatase was monitored following addition of IL-6/aptamer mixture to the HEPG2 reporter cell line. In the absence of aptamer, cells incu- bated with IL-6 gave an A of 0.3, and in cells incubated with medium alone, the A was 0.06. ADR58 inhibition of OSM-induced secreted alkaline phosphatase (M) was included as a positive control. The exper- iment was carried out in triplicate. Error bars represent S.D.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Control, Incubation, Inhibition, Positive Control

Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 9. Functional activity of truncated ADR58. a, effect of trun- cated ADR58 on OSM signaling via the OSM receptor in the HEPG2 reporter cell line, measured by production of secreted alkaline phospha- tase (f). Aptamers were preincubated with 2 ng/ml OSM, and produc- tion of secreted alkaline phosphatase was monitored following addition of OSM/aptamer mixture to the HEPG2 reporter cell line. The A from cells incubated with OSM in the absence of aptamer was 0.443, and that from cells incubated with medium alone was 0.035. b, activity of trun- cated ADR58 in OSM-gp130 ELISA (f). The ability of truncated ADR58 to block the interaction of microtiter plate-immobilized OSM with gp130 is shown. gp130-IgG-Fc fusion binding was quantitated with an alkaline phosphatase-linked anti-IgG antibody. The experiment was carried out in triplicate. Error bars represent S.D. At the concentration of gp130-IgG-Fc used, the OSM was approximately at the ED50. The A values are corrected for nonspecific binding (where either OSM or gp130-IgG-Fc is excluded; A 5 0.06). The A in the presence of OSM and gp130-IgG-Fc but without aptamer was 1.29 6 0.3.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Functional Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Binding Assay, Concentration Assay