Aspartate assay kit

The assay was conducted according to the manufacturer’s instructions for the Aspartate Assay Kit (Cat# ab102512; Abcam). PCa cells were collected by trypsinization and ultrasonic digestion (output power = 15 W, duration = 5 s, interval = 5 s, pulse number = 12) in the aspartate assay buffer provided by the kit to release the aspartate content within the cells. Low-speed centrifugation (2,500g, 10 min) removed cell debris sediments. Results were obtained by measuring in OD 570 nm.

Intracellular aspartate quantitation was performed at 0.1 and 30 MPa on cells in mid-exponential growth cultured in 100 mL bottles at the indicated hydrostatic pressures (by using the same protocol as above for the RNA preparation). Triplicate cultures were performed. Cells were pelleted via centrifugation at 9000× g for 15 min at 4 °C and then washed once with 1 mL of 200 mM NaCl. After centrifugation, the pellet was suspended with 0.5 mL hot distilled water and incubated for 15 min at 100 °C, followed by incubation on ice for 10 min. Then, the extract was centrifuged at 15,000× g for 5 min at 4 °C to eliminate cell debris, and the supernatant was aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C. Aspartate was measured by using the fluorometric Aspartate assay Kit (Abcam, Cambridge, UK). Samples were plated in 96-well black microplates (Costar^TM 96-well Assay Plates, Black Polystyrene) for fluorescence readings at Ex/Em 535 nm/587 nm on a TECAN-Spark Infinite M200 plate reader. In the meantime, the number of cells on each sample was counted as previously described [22 (link)] in order to relate the aspartate concentration to the cells amount. Experiments were performed in triplicate.

Transfected cells (after 48 h) or MEF‐BRCA1^▵/▵ and MEF‐BRCA1^+/+ cells were dissociated and counted. The intracellular levels of aspartate and α‐KG were examined by Aspartate Assay Kit (Abcam) and Alpha Ketoglutarate Assay Kit (Abcam). Briefly, 2 × 10⁶ cells were homogenized in PBS and the supernatant collected. The flow‐through containing the metabolites was used for the measurement of aspartate and α‐KG, following the manufacturer's instructions. The abundance of aspartate and α‐KG were normalized to the cell number. For mitochondrial aspartate, α‐KG and NADH/NAD⁺ redox detection, mitochondria were separated from 5 × 10⁶ transfected cells or MEF‐BRCA1^▵/▵ and MEF‐BRCA1^+/+ cells via commercial kits (Beyotime, Shanghai, China), which has been reported to separate mitochondria from cytosol (Wang et al., 2016a). The mitochondria were homogenized in PBS and the supernatant was collected. The flow‐through containing the metabolites was used for the measurement of aspartate, α‐KG and NADH/NAD⁺ redox (Beyotime), following the manufacturer's instructions.

Differentiated iBACs (day 7–8 of differentiation) were harvested in homogenization buffer (PBS pH 8.5, 1 mM DTT, 10% glycerol) and sonicated 3× for 5 s. Cell homogenates were centrifuged for 10 min/10000 × g/4 °C and supernatants were collected. An equal amount of 2× reaction buffer (dH₂O pH 8.5, TrisHCl 100 mM, NaCl 100 mM, CaCl₂ 5 mM, DTT 0.2 mM, IGEPAL CA-630 (1%, Sigma) 0.5%, N-acetylaspartic acid (Sigma) 40 mM) was added to the samples and a defined volume per sample was collected to provide the same background matrix for the aspartate standard curve. The background matrix was heated at 95 °C for 10 min and subsequently centrifuged for 3 min/16000 × g/4 °C. The background supernatant was collected and all samples and background sample were incubated for 18 h/38 °C/350 rpm in a thermomixer. After incubation, all samples were inactivated at 95 °C for 10 min and subsequently centrifuged for 3 min/16000 × g/4 °C. Supernatants were used for analyzing released aspartate content using the aspartate assay kit (BioVision).