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. 2015 Jun 1;6(26):22239–22257. doi: 10.18632/oncotarget.4161

Figure 9. Integrin inhibition differentially influences cell growth in 2-D and 3-D cultures of OPNb- and OPNc-expressing cells.

Figure 9

A. Cilengitide reduces OPNb but not OPNc cell proliferation in 2-D cultures. Representative plates were shown. OPNb, OPNib, OPNc, OPNic stable and LacZ control cells were seeded in sextuplicate at 150 cells/6-well. Sets of 3 wells were mock-treated or treated with IC50LacZ dose of cilengitide (1000 nM) for 12 days, and clonal foci were fixed and stained with Diff-Quik Staining solution. B. Bar graph representation of stained foci showing significantly reduced OPNb and OPNib cell proliferation in the presence of cilengitide as compared to their untreated cells, which exceeded the 50% (IC50LacZ) reduction observed in treated LacZ control, OPNc or OPNic cells. (***P < 0.001, ****P < 0.0001, t-test) C. Ectopic expression of both OPNb and OPNc enhanced EAC cell colony formation in 3-D soft agar cultures. Integrin inhibition did not significantly alter the colony formation of these cells. Flo/OPN and LacZ control cells (7000 cells/12-well, from the same cell-suspensions in Figure 9A) were mixed with top-agar media in the presence or absence of cilengitide (1000 nM) and were pipetted onto the casted base-agar with or without cilengitide. Cells were allowed to grow for 20 days with periodic replacement of the overlaid media with or without cilengitide. Agar plates were stained with 0.05% crystal violet in 50% ethanol and imaged using the Leica MXFL III Stereo microscope; Olympus DP-70 digital camera at × 0.8 magnification. Soft agar assays were performed in triplicate. D. Significantly increased colony-formation was observed in Flo/OPNb, OPNib, OPNc and OPNic cells as compared to Flo/LacZ controls and was not altered with the addition of cilengitide. Colonies were counted using ImageJ software, and graphs were generated using Prism software. (red, comparison to untreated LacZ; green, comparison to cilengitide-treated LacZ; ns, not significant; **P < 0.01, ***P < 0.001, ****P < 0.0001, One-way ANOVA)