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. 2013 Oct 1;8(10):e76327. doi: 10.1371/journal.pone.0076327

Figure 8. Behavior of contractolated cells in motility and invasion assays, and association with matrix metalloproteinase activity.

Figure 8

(A) The mean distance moved (in pixels), as a measurement of cell motility of pII cells after an overnight incubation in either a gassed incubator (pH 7.4, solid bar) or after a brief (1h) culture in un-gassed incubator at 37°C and subsequent overnight incubation in a gassed incubator (open bar). (B) The total number of pII cells showing random invasion (as defined in Methods) after an overnight incubation in a gassed incubator (pH 7.4, solid bar, set as 100%), after culture in un-gassed incubator at 37°C for 1-2h and subsequent overnight incubation in a gassed incubator, or after an overnight incubation in un-gassed incubator at 37°C (hatched bars). Asterisks denote significant difference from pH 7.4 with * p≤0.0001, and # p= 0.0004. (C) The total number of pII cells showing random invasion in response to EGF stimulation (50ng/ml) after either an overnight incubation in a gassed incubator (pH 7.4, set as 100%), or after a brief (1h) culture in un-gassed incubator at 37°C and subsequent overnight incubation in a gassed incubator (open bar). Asterisks denote significant difference from pH 7.4 with * p=0.0024 (D) The effect of amiloride or zoniporide (10µM) treatment of the total number of pII cells showing random invasion after an overnight incubation in a gassed incubator (pH 7.4, open bars), or after a brief (1h) culture in un-gassed incubator at 37°C and subsequent overnight incubation in a gassed incubator (solid bars). Asterisks denote significant difference from pH 7.4 (no drug) with * p=0.01 (E) Effect of pH and EGF stimulation on general MMP activity in pII cells. Cells were either cultured in a gassed incubator (pH 7.4) or in un-gassed incubator for 1h and either left untreated or stimulated with EGF (50ng/ml) for 30 min and metalloproteinase activity was determined using a fluorogenic substrate as described in Methods. Asterisks denote significant difference from no EGF treatment groups with * p≤ 0.05. (F) Effect of pH, EGF, amiloride, and zoniporide treatment of MMP2/9 activity in pII (open bars) and YS1.2 (solid bars) cells. Cells were either cultured in a gassed incubator (pH 7.4) or in un-gassed incubator for 1h and either left untreated, treated with amiloride or zoniporide (10µM, 60 min), or stimulated with EGF (50ng/ml, 30 min) and MMP2/9 activity was determined using a fluorogenic substrate as described in Methods. Asterisks denote significant difference from pH 7.4 with * p≤0.001, from pH7.4 +EGF with # p≤0.0001, and from alkaline pH (1h) with ** p ≤0.001. Bars represent means ± SEM for at least 3 independent determinations.