Figure 6.
Matriptase activation of PAR-2 increases MMP9 expression to mediate a loose spheroid phenotype. (A) ES-2 and NCI/ADR-Res Vec and Mat hanging drop spheroids were grown in the presence of vehicle control (DMSO) or MMP inhibitor GM6001 (50 μM) for 4 d. Graphs show the quantitation of the spheroid morphology (% loose spheroids and % empty space) as described in the Materials and methods. Data represents the average of three independent experiments (error bars are ±SEM; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, ns = not significant). (B) mRNA from ES-2 and NCI/ADR-Res Vec and Mat cells was collected and analyzed for MMP14, MMP2, and MMP9 expression by qPCR. mRNA expression was normalized to GAPDH and represented as average fold change relative to Vec (error bars are ± SEM from at least three independent experiments performed in triplicate; *P < 0.05, **P < 0.01). (C) ES-2 and NCI/ADR-Res Vec and Mat hanging drop spheroids were grown in the presence of vehicle control (DMSO) or MMP9 inhibitor JNJ 0966 (10 μM). Representative images at 4× magnification are shown in top panels; scale bars represent 650 μm. Graphs below the images show the quantitation of the spheroid morphology (% loose spheroids and % empty space) as described in the Materials and methods. Data represents the average of three independent experiments (error bars are ± SEM **P < 0.01, ***P < 0.005, ****P < 0.001, ns = not significant). (D) mRNA from ES-2 or NCI/ADR-Res Vec and Mat hanging drop spheroids treated with vehicle control (DMSO) or GB-83 (5 or 25 μM, respectively) was collected and analyzed by qPCR for MMP9 mRNA expression. mRNA expression was normalized to GAPDH and represented as average fold change relative to Vec from three independent experiments performed in triplicate (error bars are ± SEM; *P < 0.05, **P < 0.01, ns = not significant). All P values were calculated according to two-tailed unpaired Student’s t tests.

Matriptase activation of PAR-2 increases MMP9 expression to mediate a loose spheroid phenotype. (A) ES-2 and NCI/ADR-Res Vec and Mat hanging drop spheroids were grown in the presence of vehicle control (DMSO) or MMP inhibitor GM6001 (50 μM) for 4 d. Graphs show the quantitation of the spheroid morphology (% loose spheroids and % empty space) as described in the Materials and methods. Data represents the average of three independent experiments (error bars are ±SEM; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, ns = not significant). (B) mRNA from ES-2 and NCI/ADR-Res Vec and Mat cells was collected and analyzed for MMP14, MMP2, and MMP9 expression by qPCR. mRNA expression was normalized to GAPDH and represented as average fold change relative to Vec (error bars are ± SEM from at least three independent experiments performed in triplicate; *P < 0.05, **P < 0.01). (C) ES-2 and NCI/ADR-Res Vec and Mat hanging drop spheroids were grown in the presence of vehicle control (DMSO) or MMP9 inhibitor JNJ 0966 (10 μM). Representative images at 4× magnification are shown in top panels; scale bars represent 650 μm. Graphs below the images show the quantitation of the spheroid morphology (% loose spheroids and % empty space) as described in the Materials and methods. Data represents the average of three independent experiments (error bars are ± SEM **P < 0.01, ***P < 0.005, ****P < 0.001, ns = not significant). (D) mRNA from ES-2 or NCI/ADR-Res Vec and Mat hanging drop spheroids treated with vehicle control (DMSO) or GB-83 (5 or 25 μM, respectively) was collected and analyzed by qPCR for MMP9 mRNA expression. mRNA expression was normalized to GAPDH and represented as average fold change relative to Vec from three independent experiments performed in triplicate (error bars are ± SEM; *P < 0.05, **P < 0.01, ns = not significant). All P values were calculated according to two-tailed unpaired Student’s t tests.

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