Elisa reader

Cell proliferation rate was assayed using CCK-8 (Dojindo). 1×10⁴ BM-MSCs were seeded on 96-well culture plates (Corning, NY, USA) and treated with THS at indicated concentrations for 24 h. OD value at 450 nm was evaluated using an ELISA Reader (Promega, Madison, WI, USA) following the manufacturer's instructions.

DNA synthesis was assessed using an enzyme-linked immunosorbent assay-based and colorimetric BrdU assay (Roche Diagnostics). AMACR-knockdown or shLacZ control GIST882 and GIST48 cells were plated into a 96-well plate at density of 3000 cells per well, and DNA synthesis was evaluated at 24, 48, and 72 h. After incubation with BrdU for 3 hours at 37°C under 5% CO₂, the labeling medium was removed, followed by fixation and final incubation with anti-BrdU-POD solution. The absorbance of the samples was measured using an ELISA reader (Promega) at 450 nm, with the absorbance at 690 nm as reference.

DNA synthesis was assessed using an enzyme-linked immunosorbent assay-based and colorimetric BrdU assay (Roche Diagnostics) as previously described [21 (link)]. GIST48 cells transduced with shPLCB4, shYAP1, or shLacZ control were plated into a 96-well plate at density of 3000 cells per well, and DNA synthesis was evaluated at 24, 48, and 72 h. The absorbance of the samples was measured using an ELISA reader (Promega) at 450 nm, with the absorbance at 690 nm as reference.

DNA synthesis was measured using an enzyme-linked immunosorbent assay (ELISA)-based and colorimetric bromodeoxyuridine (BrdU) assay (Roche Holding AG, Basel, Switzerland). MAP1B-knockdown or shLacA control RTCC1 and J82 cell lines were plated into a 96-well plate at a density of 3000 cells per well. At 24, 48, and 72 h, we measured the amount of DNA synthesis. The labeling medium was removed after three hours of incubation with BrdU at 37 °C under 5% CO₂, followed by fixation and a final incubation with an anti-BrdU-POD solution. An ELISA reader (Promega Corp., Madison, WI, USA) was used to measure the absorbance at 450 nm, and the reference was set at an absorbance of 690 nm.

The colorimetric 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay (Sigma-Aldrich, St. Louis, MO, USA) was used to assess cell viability as previously described [30 (link)]. Vinblastine sulfate (Hospira UK Ltd., Maidenhead, UK) was obtained and suspended in normal saline. RTCC1 and J82 cells were seeded in 96-well plates at a density of 5 × 10³ cells per well the day before treatment at the indicated time points with vehicle control (0.9% saline) or increasing concentrations of vinblastine sulfate. The length of treatment interval was 72 h. After incubation with XTT reaction mixture for three hours at 37 °C under 5% CO_2, the absorbance of the samples was determined using an ELISA reader (Promega Corp., Madison, WI, USA) at 450 nm, with the absorbance set at 630 nm as reference.

The generation of ROS during subtilisin infiltration was determined using a commercial plant ROS enzyme-linked immnunosorbent assay (ELISA) kit (Chundubio). Three or four upper leaves without infiltration of N. benthamiana were harvested at various times after infiltration with subtilisin, 0.1 g was fully ground in liquid nitrogen, and phosphate-buffered saline (pH 7.4) was added. The plant tissue was homogenized with a low-temperature homogenizer, centrifuged twice for about 20 min, and then the precipitate was discarded for use in ELISA. The standard curve was used to determine the amount in each unknown sample. OD₄₅₀ values were measured with an ELISA reader (Promega). Meanwhile, ROS generation was detected using 3,3′-diaminobenzidine (DAB) solution (Solarbio) as described previously (Yang et al., 2018 (link)). Leaves were then decolorized in boiling ethanol (90%) for 30 min and were photographed by camera (Zhao et al., 2018 (link)).