ES-2 and ES-2:ES-2-Rv-NICD3 cells were plated at 2 × 105 cells/well in 6 well plates and cultured 48 h before treatment with the hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU, 1 mM, Sigma-Aldrich) or vehicle control (PBS) for 24 h [37 (link)]. Protein lysates from monolayer and spheroids were prepared in RIPA buffer as described previously [59 (link)]. ES-2, A2780 and OVCAR3 cells were cultured to 80% confluency before protein isolation. 20 µg of protein was electrophoresed on 4–20% TGX gels (Bio-Rad, Hercules, CA, USA) and transferred overnight to PVDF membrane (GE healthcare, little Chalfont, England). Proteins were detected with DAB2 rabbit monoclonal antibody (1/2000, ab256524, Abcam), anti-rabbit IgG peroxidase conjugated antibody (1/4000, Sigma-Aldrich) and Amersham™ ECL™ Prime (Cytvia, Marlborough, MA, USA). Chemiluminescence was detected using the ChemiDoc™ Imaging System (Bio-Rad) and band intensity was calculated with ImageLab software (Bio-rad, version 6.1). β-actin monoclonal mouse antibody (1/5000, ab8226, Abcam) or GAPDH monoclonal mouse antibody (1/50,000, 60004-1-Ig, Proteintech®, Rosemont, IL, USA) was used as a loading control.
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