Figure 5.
Matriptase activation of PAR-2 triggers PI3K/Akt signaling to mediate loose spheroid morphology. (A) ES-2 Vec or Mat cells were treated with vehicle control (DMSO), 10 μM U0126 (MEK1/2 inhibitor), 10 μM SB202190 (p38 MAPK inhibitor), or 10 μM LY294002 (PI3K inhibitor) and allowed to form hanging drop spheroids 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). Right: Representative images of ES-2 Vec and Mat hanging drop spheroids treated with vehicle control (DMSO) or LY294002 are shown at 4× magnification; scale bars represent 650 μm. (B) NCI/ADR-Res Vec or Mat spheroids were treated with vehicle control (DMSO) or LY294002 (10 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, ns = not significant). Right: Representative images at 4× magnification are shown; scale bars represent 650 μm. (C) Whole-cell lysates from ES-2 (left panels) and NCI/ADR-Res Vec and Mat (right panels) hanging drop spheroids treated with vehicle control (DMSO), GB-83 (5 μM for ES-2 and 25 μM for NCI/ADR-Res) or LY294002 (10 μM) were collected with RIPA lysis buffer, analyzed by SDS-PAGE and immunoblotted with rabbit anti-human p-Akt (Ser473), total Akt, and β-tubulin. GB-83 slightly affected total Akt levels in both ES-2 Mat and Vec spheroids. Densitometric analysis was conducted with ImageJ and reported as relative densitometric units normalized to β-tubulin and compared to Vec; average of three independent experiments (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. Source data are available for this figure: SourceData F5.

Matriptase activation of PAR-2 triggers PI3K/Akt signaling to mediate loose spheroid morphology. (A) ES-2 Vec or Mat cells were treated with vehicle control (DMSO), 10 μM U0126 (MEK1/2 inhibitor), 10 μM SB202190 (p38 MAPK inhibitor), or 10 μM LY294002 (PI3K inhibitor) and allowed to form hanging drop spheroids 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). Right: Representative images of ES-2 Vec and Mat hanging drop spheroids treated with vehicle control (DMSO) or LY294002 are shown at 4× magnification; scale bars represent 650 μm. (B) NCI/ADR-Res Vec or Mat spheroids were treated with vehicle control (DMSO) or LY294002 (10 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, ns = not significant). Right: Representative images at 4× magnification are shown; scale bars represent 650 μm. (C) Whole-cell lysates from ES-2 (left panels) and NCI/ADR-Res Vec and Mat (right panels) hanging drop spheroids treated with vehicle control (DMSO), GB-83 (5 μM for ES-2 and 25 μM for NCI/ADR-Res) or LY294002 (10 μM) were collected with RIPA lysis buffer, analyzed by SDS-PAGE and immunoblotted with rabbit anti-human p-Akt (Ser473), total Akt, and β-tubulin. GB-83 slightly affected total Akt levels in both ES-2 Mat and Vec spheroids. Densitometric analysis was conducted with ImageJ and reported as relative densitometric units normalized to β-tubulin and compared to Vec; average of three independent experiments (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. Source data are available for this figure: SourceData F5.

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