Skip to main content
. 2017 Feb 24;8(23):37061–37079. doi: 10.18632/oncotarget.15715

Figure 4. In vitro studies to determine 3C23K mechanisms of action.

Figure 4

(A) Clonogenic assay: Clonogenic survival of COV434-WT and COV434-MISRII cells incubated or not (medium) with MIS (0.1 or 50 nM) or 3C23K (1 or 100 μg/ml). After 15 days of culture in the presence of MIS or 3C23K, colonies were fixed with a methanol/acetic acid solution (3:1), stained with 10% Giemsa and counted. (B) Smad1/5 phosphorylation: Western blot analysis of serum-deprived (for 48 hours) COV434-MISRII (left panel) or NIH-OVCAR-3 (right panel) cell extracts obtained after incubation or not with MIS l (0.1, 1, 10 or 50 nM) for 1 hour using anti-phospho-Smad1/5 (Ser463/465) (41D10) rabbit MAb. MIS induced Smad1/5 phosphorylation in MISRII-overexpressing COV434 and NIH-OVCAR3 cells at all the tested concentrations in a dose-dependent manner. (C) Apoptosis: After incubation with 50μg/ml MAb (as indicated) or 150 nM staurosporin (positive control) for 24 hours, COV434-MISRII cells were stained using the Annexin V-FITC Apoptosis Detection Kit (Beckman Coulter IM3614). Results of one representative experiment out of four are shown and are expressed as the percentage of cell labeled with Annexin V, propidium iodide (PI) or both. (D and E) ADCC: COV434-MISRII cells were incubated with human NK cells (E:T ratio = 10) purified from healthy donors’ peripheral blood (D) or human, cynomolgus monkey or mouse PBMC (E:T ratio indicated in the figure) (E) and increasing concentrations of antibody (3C23K, 32C3K-CHO or irrelevant MAb) at 37°C for 4 hours. The lysis of target cells was assessed by quantifying the release of lactate dehydrogenase (LDH) by target cells in the supernatant and calculated according to the formula: % lysis = [(ER-SR)/(100-SR)]-[(NC-SR)/(100-SR)], where ER, SR and NC represent the experimental LDH release, the spontaneous LDH release (target cells without NK cells and without antibody) and the natural cytotoxicity (target cells + NK cells without antibody), respectively. (F) ADPC: COV434-MISRII target cells labeled with the CMFDA dye (CellTracker™Green, Life Technologies) and pre-incubated with increasing concentrations of antibody (3C23K or irrelevant MAb) at room temperature for 30 minutes were mixed (10:1 E:T ratio) with macrophages derived from monocytes obtained from human or cynomolgus monkey PBMC or mouse bone marrow. Living target cells were quantified by flow cytometry after 3 (human and cynomolgus monkey macrophages) or 5 days (mouse macrophages). Results are expressed as percentages relative to the corresponding isotype control.