Cell growth assays

OVCAR3 cells with Dox-inducible knockdown and re-expression of RACK1 were plated at a density of 2,000 cells per well in a 96-well plate in growth medium containing 0.5 μg/mL puromycin, 200 μg/mL G418, and 1 μg/mL Dox. Twenty-four hours after plating the cells, the growth medium was replaced with fresh medium supplemented with 0.5 μg/mL puromycin, 200 μg/mL G418, and 1 μg/mL Dox in the presence of vehicle or 3 nM thapsigargin. The cells were grown for the indicated amount of time. At the end of the indicated times, cells were fixed with 4% paraformaldehyde for 15 minutes, washed with water, and stored at 4°C. The fixed cells were then stained with crystal violet (0.5% crystal violet in 20% methanol) for 30 minutes with gentle agitation at room temperature. The stained cells were washed with water and air dried. The crystal violet was then dissolved in 10% acetic acid and the absorbance at 590 nm was measured using a spectrophotometer. The absorbance of a blank well was subtracted from the samples and the values were normalized to the values at Day 0. Three independent biological replicates were performed to ensure reproducibility. Statistical differences were determined using two-way ANOVA.

Ovarian cancer cells were plated at a density of 2,000 cells per well in 96-well plates. Twenty-four hours later, the cells were treated with single or combined treatments of 10 μM PARP14 inhibitor and 3 nM thapsigargin for the indicated amount of time. At the end of the indicated times, the cells were processed for the crystal violet staining assay as described above.