Mars data analysis computer software

This was performed as described previously [19 (link), 31 (link)]. Cells (3X10⁴) were seeded and transfected in 96-well plates on the same day and after 24 h, cell culture medium was replaced. Chemotherapy (paclitaxel or cisplatin) at different concentrations (0 to 320 μg/ml) was added to cells for 48 h after which culture medium was replaced and 100 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) solution (Sigma-Aldrich) dissolved in 1x PBS solution (0.5 mg/ml, final concentration) (Sigma-Aldrich) was added to the cells. After 2 h incubation, cell culture medium was replaced with 100 μl of dimethyl sulfoxide (DMSO). Absorbance was read at OD595nm using the CLARIOstar Plate Reader (BMG Labtech, Germany) and MARS Data Analysis Computer Software (BMG Labtech, Mornington, Victoria, Australia).

MTT assay is a colorimetric assay used to quantify the survival of viable cells (38 (link), 39 (link)). In brief, 3 x 10⁴ cells were seeded into 96-well plates overnight. Cell lines were treated with either paclitaxel (PTX) (EbeweR, SANDOZ, Novartis, Basel Switzerland) or cisplatin (CIS) (Accord Healthcare Pty Ltd, Melbourne, Australia) and MTT assay was performed by replacing culture media with 100µL of thiazolyl blue tetrazolium (MTT) solution (Sigma-Aldrich) dissolved in 1x PBS solution (0.5mg/mL final concentration) (Sigma-Aldrich). Cells were incubated for 2 hours at 37°C in 5% CO₂ humidity. Media was discarded and replaced with 100µL of dimethyl sulfoxide (DMSO). Absorbance was read at OD595nm using the CLARIOstar Plate Reader (BMG Labtech, Germany) and data was analysed by MARS Data Analysis Computer Software (BMG Labtech, Mornington, Victoria, Australia).

This was performed as described previously [16 (link), 24 (link)]. Control, siRNA, and CRISPR/Cas9 TIMP-2 knocked down OVCAR5 cells were either left untreated or treated with paclitaxel (PTX) at varying concentrations (0 to 320 µg/mL) for 48 h. For puromycin cell death, OVCAR5 cells were incubated for 48 h in different puromycin concentrations ranging from 0 nM to 320 µg/mL. The culture medium was replaced with 100 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) solution (Sigma-Aldrich) dissolved in 1× PBS solution (0.5 mg/mL, final concentration) (Sigma-Aldrich). After 2 h incubation, the MTT solution was replaced with 100 µL of dimethyl sulfoxide (DMSO). Absorbance was read at OD595nm using the CLARIOstar Plate Reader (BMG Labtech, Germany) and analysed by MARS Data Analysis Computer Software (BMG Labtech, Mornington, Victoria, Australia). Each concentration was repeated in quadruplicates and each experiment was repeated 3 times.