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Patients and Samples (Primary Cell Culture)
This study was conducted according to the “REporting recommen-dations for tumor MARKer prognostic studies” . To establish primary, patient-derived ovarian cancer cell cultures, we analyzed samples from patients undergoing surgical resection between January 2015 and March 2018 at the Department of Gynecology of the Goethe University Hospital in Frankfurt am Main, Germany. For the samples with validated diagnosis, sufficient archival material for immunohistochemical analysis was available. The Local Research Ethics Committees approved studies of human tissue, and samples were processed anonymously.
Antibodies and Chemicals
Western Blot Analysis
Protein extracts of cells were prepared by lysis in RIPA buffer (Sigma) supplemented with protease inhibitors (Complete protease inhibitor cocktail, Roche). Protein extracts (25 μg) were separated by SDS-PAGE and transferred onto PVDF membranes using the TransBlot Turbo Transfer System (BioRad). After blocking with 5% BSA in PBS with 0.1% Tween-20 for 30 minutes, the membrane was incubated with primary Fasudil for 1 hour at room temperature. HRP-linked secondary antibodies were incubated 30 minutes at room temperature followed by ECL detection (ECL Chemiluminescent Western Blot Substrate, Pierce).
Colony Formation Assay
Cells were treated with paclitaxel, BI6727, or both for 24 hours. In case of combinatorial treatments with proTAME, cells were treated first with paclitaxel/BI6727 for 24 hours followed by proTAME for 24 hours. Subsequently, 2000 cells were seeded in 6-well plates. Colonies were fixed using 70% EtOH and stained with Coomassie Brilliant Blue. The numbers of grown colonies were counted and images were taken using AxioObserver Z1 microscope (Zeiss) as well as the ChemiDoc MP system (BioRad).
The ovarian carcinoma cell lines OVCAR-3 and SKOV-3 were cultured in RPMI 1640 (Gibco) and in McCoy's 5a (Gibco), respectively, both containing 10% FCS (Gibco) and 1% Penicillin/ Streptomycin (Sigma-Aldrich). Primary cells were isolated from ovarian cancer tissues using the tumor dissociation kit (Max Miltenyi 130-095-929) together with the tumor cell isolation kit (Max Miltenyi 130-108-339) following the manufacturer's instructions. Isolated fibroblasts were cultured as described .
Three-Dimensional (3D) Cultures
A cell suspension of 3000 cells/50 μl was prepared and pipetted from the topside into a 96-well Perfect 3D Hanging Drop plate (BioTrend). Plates were incubated at 37°C for 3 days until hanging drops have developed. The 3D culture was harvested on a 96-well plate covered with 1% agarose by low-spin centrifugation. Treatment of cells with BI6727, paclitaxel, and/or proTAME was performed as indicated. Cells were stained with the LIVE/DEAD viability/cytotoxicity kit (Molecular Probes/Thermofisher) for 30 minutes and inspected using a fluorescence microscope. While the polyanionic dye calcein is retained in live cells, producing an intense uniform green fluorescence, EthD-1 enters cells with damaged membranes, thereby producing a bright red fluorescence upon binding to nucleic acids in dead cells. The ratios of viable/dead cells were calculated with the software ImageJ Fiji.
for further 3 hours at 37°C/5% CO2 before fluorescence reading (Victor X4, PerkinElmer). The activity of Caspase-3/7 was determined using the Caspase-Glo 3/7 Assay (Promega). Twenty microliters of substrate per well was applied, and after 30-minute shaking at room temperature in the dark, luminescence was detected (Victor X4, Perkin Elmer). The analysis of data was done online by Networkanalyst and GraphPad Prism.
Cell Cycle and Apoptosis Assays
The treatment with paclitaxel, BI6727, and/or proTAME was conducted at least for 24 hours followed by cell cycle and apoptosis measurements after predefined time intervals. For cell cycle analysis, cells were harvested, washed, fixed with 70% EtOH, and stained as described [45,46]. Cell cycle quantification was performed using a FACS Calibur and Cellquest Pro software (both BD Biosciences). The activity of Caspase-3/7 was determined in cell lysates using the Caspase-Glo 3/7 Assay according to the manufacturer's instructions.
To measure cell proliferation using the Cell Titer-Blue Cell Viability Assay, 2500 cells per well were seeded in 96-well plates and treated with paclitaxel, BI6727, and/or proTAME. Cells were incubated with 20 μl substrate of the Cell Titer-Blue Cell Viability Assay for 4 hours, and the light absorbance was measured at 540 nm (Victor X4, Perkin Elmer). The 50% inhibitory concentration (IC50) was estimated using the following formula: 100 × (T − T0)/(C − T0) = 50, where T is the optical density (OD) value after drug treatment, T0 is the OD value at time 0, and C is the OD value for the diluent treatment. Time 0 was defined as the day the drug was administered.