Filtermax f3

We used the PRDX1 ELISA kit (Abnova, Cat. No. KA0536) for the quantitative determination of human PRDX1 in the plasma samples. This assay is based on a sandwich ELISA. The 96-well plates provided were pre-coated with a monoclonal antibody specific to human PRDX1. Briefly, 100 μL of standards for calibration curves and homemade standards (in duplicates), and plasma samples (single analysis) diluted 1:5 were added to each well and the plates were incubated for 1 hour at room temperature followed by three washes with 300 μL of 1X wash buffer. Next, 100 uL of 1X “working AV-HRP solution” was added to each well and the plates were incubated for 30 minutes at room temperature followed by three washes with 300 μL of 1X wash buffer. Finally, 100 μL of substrate were added to each well turning the liquid blue. The plates were incubated for 10 minutes at room temperature. To stop the reaction, 100 μL of solution stop were added to each well turning the liquid yellow. The absorbance was measured at 450 nm within 20 minutes after the addition of the stop solution on the microplate reader FilterMax F3, Molecular Devices. All measurements were blinded. The reproducibility of the assay was assessed from standards by determining intra- and inter-run coefficients of variation.

AGS, AGSR, MKN-74, and MKN-74R cells (1 × 10⁵ cell/well) were seeded in a 6-well plate and transfected with 2 μg pGL2 luciferase vector (Promega, Madison, WI, USA) and reporter luciferase vector containing an E-cadherin promoter (−368~+51) after 24 hours using Lipofectamine 2000 agent (Invitrogen, Carlsbad, CA, USA). Luciferase activity was measured using a dual-luciferase reporter assay kit (Promega, Madison, WI, USA) on Molecular Devices Filter Max F3 (Molecular Devices, CA, USA). The luciferase activity was normalized to the activity of the Renilla luciferase.

MTS viability assays were described previously [41 (link), 58 (link)]. In brief, 2.5 × 10³ GSCs were plated in a 96-well plate. Cells were then treated with DMSO, 50 μM of TMZ, 100 μM of αCT1, or a combination of TMZ and αCT1. αCT1 treatment was repeated every fourth day for 2 doses. In another set of experiments, cells were treated with imatinib or γ-secretase inhibitor IX as described above. After one week, cell viability was monitored using the MTS assay. 10 μl of MTS (Promega) was added to each well, then incubated at 37°C for 1 h. The absorbance at 490 nm was measured using a FilterMax F3 microplate reader (Molecular Devices, LLC) according to manufacturer's instructions. Percent cell viability was obtained by dividing the absorbance of treatment groups with those of untreated groups.

The phosphatase assay was conducted with the EnzCheck Phosphatase Assay Kit (Molecular Probes, Eugene, OR, USA). The 100 μL reaction mixture in the 96-well black plate for the measurement of phosphatase activity consisted of a 100 μM 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) substrate, 1 mM metal such as Mg²⁺, Zn²⁺, Mn²⁺, or Ca²⁺, 100 mM NaCl, 25 mM Tris-HCl pH 7.0, and a 10 μM protein sample. The reaction was started by adding the protein, and the results were measured with the Filter Max F3 (Molecular Devices, San Jose, CA, USA) as soon as possible after adding the protein to the reaction mixture. The interval of measurement was 3 min. The amount of the product, 6,8-difluoro-4-methylumbelliferyl (DiFMU), in the solution was determined photometrically at 460 nm. The graph for the phosphatase assay was modified using Origin 2022 (OriginLab, Northampton, MA, USA). The data were fitted to the quadratic equation to calculate a Kd value between Mg²⁺ and Sav2152, as follows: $$\mathrm{V}={\mathrm{V}}_{\mathrm{m}\mathrm{a}\mathrm{x}}\times \frac{\left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\left[{\mathrm{A}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\mathrm{K}\mathrm{d}-\sqrt{{\left(\left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\left[{\mathrm{A}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]+\mathrm{K}\mathrm{d}\right)}^2-4\times \left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]\times \left[{\mathrm{A}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]\times \mathrm{K}\mathrm{d}}}{2\left[{\mathrm{P}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}\right]}$$
where V represents the initial reaction velocity, V_max represents the maximum initial reaction velocity, P_total represents the total amount of protein, A_total represents the total amount of Mg²⁺ ion, and Kd represents a dissociation constant.

Cells (1 × 10⁶ cells/well) were cotransfected with 2.5 μg of human COX-2 promoter luciferase constructs and pRL-renilla vectors (Promega, Madison, WI), according to the manufacturer’s protocol, by using the Neon™ transfection system. Luciferase activities were measured consecutively by using the Dual Luciferase Assay System (Promega) with a FilterMax F3 microplate reader (Molecular Devices, CA).