Absorbance reader

Cell Proliferation assays were performed on cells using a cell titer 96 (MTS) aqueous reagent (Promega, Madison, WI, USA). 2 × 10³ cells were seeded in a 96-well plate, 12 h before the experiment. Then the cells were divided into groups of NC, siRNA1-PSMB2(S1) and siRNA2-PSMB2(S2) and incubated for 0, 24, 48, 72 or 96 h. MTS solution added to each well and incubated for 30 min at 37 ℃. Absorption of triplicate samples was measured at 490 nm using an absorbance reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The effect of A939572 on cell proliferation was assessed by counting viable cells using a colorimetric assay in the Cell Counting Kit‐8. Cells were seeded in 96‐well plates at 2000 cells per well for 24 hours, and A939572 was then added at concentrations of 0, 3.2 and 25.6 µg/mL for 24 hours. The absorbance at 450 nm was measured by an absorbance reader (Bio‐Rad).

In vitro cytotoxicity of the platinum compounds against human hepatocellular carcinoma (HepG-2), gastric carcinoma (SGC-7901), non-small-cell lung cancer (A549), and colorectal cancer (HCT-116) cell lines were measured by the MTT assays. Briefly, the cells were seeded in 96-well cultured plates at a density of 5000 cells/well. After overnight incubation (16 h), the cells were treated with the platinum complexes. After 48 h of incubation, 10 μL of a freshly diluted 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (5 mg/mL) were added to each well and the plates were incubated at 37 °C in a humidified 5% CO₂ atmosphere for 4 h. At the end of the incubation period the medium was removed and the formazan product was dissolved in 150 μL of DMSO. The cell viability was evaluated by measurement of the absorbance at 490 nm, using an Absorbance Reader (BioRad). IC₅₀ values (compound concentration that produces 50% of cell growth inhibition) were calculated from curves constructed by plotting cell survival inhibitory rate (%) versus drug concentration logarithm. All experiments were repeated in three times. The reading values were converted to the percentage of control (% cell survival). Cytotoxic effects were expressed as IC₅₀ values.

Intracellular ATP content was quantified with the enhanced ATP assay kit according to the user manual (Beyotime). After cells (5 × 10⁵ per test) were lysed, ATP concentration in the supernatant was quantified by measuring the fluorescence emitted by firefly luciferase on a luminometer (Berthold, Germany). The concentration of intracellular ATP was shown in nmol/mg protein. Total protein was quantified for normalization using the Bio-Rad Protein Assay (Bio-Rad).
Citrate concentration was determined with a citrate assay kit following the manual (Abcam, Cambridge, UK). Briefly, the assay buffer was used to collect cells (2 × 10⁶ per test), and supernatants were incubated with 50 μL citrate reaction mixture containing an enzyme mix, the developer and the citrate probe for 30 min at room temperature. Absorbance was measured at 570 nm on an Absorbance Reader (Bio-Rad). Citrate concentration in the supernatant was extrapolated from a standard curve and normalized to cell number. Each assay was performed in at least three technical replicates of independent experiments.

The PCa/macrophage cells were co-cultured in 0.4 μM pore size transwell plates for 24 hours. Then the conditioned media (CM) were collected, diluted with normal culture media, plated into the lower chamber of new 24-well transwells with 5 μM pore polycarbonate membrane insert (Corning, #3422, BD Biosciences, San Jose, CA). 1×10⁵ of parental THP-1 cells were plated onto the upper chamber for macrophage migration assay. The cells migrated into the lower chamber were measured using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)method. MTT solution (100 mL, 5 mg/mL in PBS) was added to each well of the transwell plates and incubated for 2 hours at 37°C. Centrifuged at 3000 rmp for 20 minutes, discarded the supernatant discarded and 300 μl/well dimethylsulfoxide (DMSO) was added to dissolve the formazan. The absorbance was immediately measured at 570 nm using a Bio-Rad Absorbance Reader. Each sample was assayed in triplicate and the experiments were repeated at least three times.

According to other studies, CPS1 not only participates in urea metabolism but also affects pyrimidine synthesis.15, 16 As ammonia was utilized by CPS1 to produce carbamoyl phosphate, the inhibition of CPS1 expression may increase the ammonia level.17 Cells were transfected with siRNA and treated as described in the ammonia assay kit (Sigma) and QuantiChrom Urea Assay Kit (BioAssay Systems) protocols. The absorbance at 450 nm was measured by an absorbance reader (Bio‐Rad).