Micro bicinchoninic acid bca assay

After the mice were euthanized, joint tissues were collected from mice in the CIA model, and lung tissues were obtained from mice in the HDM-challenge model, as described above. The tissues were flash frozen in liquid nitrogen and stored in −80°C until further use. The flash frozen tissues were homogenized on ice using a tissue homogenizer (Omni International, USA), in protein extraction buffer T-PER (Thermo Scientific, USA) containing protease inhibitor cocktail (Cell Signaling Technology, Denver, USA). The homogenates were centrifuged at 10,000 × g, at 4°C for 10 min. The supernatants were collected, aliquoted and stored in −20°C until use. Total protein amount was estimated in the supernatants using micro-Bicinchoninic acid (BCA) assay (Thermo Scientific, USA) according to the manufacturer's instructions.

OV90 and OVCAR8 cells were grown in as spheroids for 3 days in TEM, and treated with disulfiram and the NCTs as indicated. Whole cell lysates were collected at the indicated time points post treatment, by pelleting spheroids and removing growth medium and washing in PBS. Lysates were collected in lysis buffer: RIPA buffer (Thermo Scientific, Waltham, MA) containing 1× protease inhibitor (Halt, Thermo Fisher Scientific, Waltham, MA) 1× phosphatase inhibitor (Phos-STOP, Millipore Sigma, Burlington, MA). After a brief incubation on ice, the lysates were homogenized by passing the samples through 26-G needles for 5 strokes, followed by centrifugation at 16,000g, 4°C for 20 min to collect the supernatant. Protein concentration was quantified by microbicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA). Lysates (40 µg) were separated by SDS-PAGE under reducing conditions, transferred onto PVDF membranes, and blocked in 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST). Membranes were incubated with primary antibodies diluted in 1% non-fat milk in TBST overnight at 4°C, washed with TBST, and then incubated with secondary HRP-conjugated mouse or rabbit IgG as appropriate. Images were generated using the Odyssey system and software (LI-COR Biosciences, Lincoln, NE).