Matriptase:HAI-1 ratios and effects on cell surface serine protease activity and spheroid phenotype across OvCa cell lines
| Cell line . | Relative Matriptase:HAI-1 protein ratioa . | Cell surface serine protease activity (fold change ± SEM)b . | Spheroid morphology . | |
|---|---|---|---|---|
| Spheroid phenotypec . | % empty space (rel. to IOSE)d . | |||
| IOSE397 | 1.0 | 1.00 | Tight | 1.00 |
| ES-2 | 3.2 | 3.19 (±0.14) | Tight | 1.27 |
| NCI/ADR-Res | 6.6 | 6.70 (±0.88) | Tight | 1.86 |
| OVCAR3 | 704.9 | 20.64 (±0.10) | Loose | 4.56 |
| CAOV3 | 144.6 | 11.94 (±1.93) | Loose | 4.60 |
| COV362 | 98.5 | 38.98 (±3.78) | Loose | 6.43 |
| Cell line . | Relative Matriptase:HAI-1 protein ratioa . | Cell surface serine protease activity (fold change ± SEM)b . | Spheroid morphology . | |
|---|---|---|---|---|
| Spheroid phenotypec . | % empty space (rel. to IOSE)d . | |||
| IOSE397 | 1.0 | 1.00 | Tight | 1.00 |
| ES-2 | 3.2 | 3.19 (±0.14) | Tight | 1.27 |
| NCI/ADR-Res | 6.6 | 6.70 (±0.88) | Tight | 1.86 |
| OVCAR3 | 704.9 | 20.64 (±0.10) | Loose | 4.56 |
| CAOV3 | 144.6 | 11.94 (±1.93) | Loose | 4.60 |
| COV362 | 98.5 | 38.98 (±3.78) | Loose | 6.43 |
Matriptase and HAI-1 protein expression were determined by immunoblot analysis; levels were quantitated by densitometric analysis, normalized to β-tubulin, and the matriptase:HAI-1 ratio was calculated (see Fig. S1 B). The data is expressed relative to control immortalized IOSE397 cells, run on the same gels.
AEBSF-sensitive cell surface serine protease activity was measured using Boc-QAR-AMC fluorogenic peptide. Fold change of endpoint fluorescent values (normalized to cell number) was calculated relative to IOSE397 cells.
Spheroids were allowed to form on nonadhesive agarose hydrogels overnight, visualized using the EVOS microscope, and qualitatively scored as tight or loose as defined in the Materials and methods.
As a quantitative measure of spheroid morphology, the percentage empty space in EVOS images of spheroids was determined using ImageJ as described in the Materials and methods and expressed relative to IOSE397 cells.