Powerscan 4

NAA and DEX treatments were performed by submerging plants into half-strength Gamborg’s B5 liquid medium [81 (link)] containing 10 μM NAA and/or 10 μM DEX for 12 h. GUS activity was then measured by monitoring cleavage of the β-glucuronidase substrate 4-methylumbelliferyl β-D-glucuronide (MUG) as described previously [29 (link)] with some modifications. After adjusting the concentration of extracted protein to 10 μg/70 μl extraction buffer, 20 μl of methanol and 10 μl of 10 mM MUG were added. After incubation at 37°C for 30 min, 900 μl of 200 mM sodium carbonate was added to stop the reaction. Fluorescence (460 nm emission/360 nm excitation) of liberated 4-methylumbelliferone (MU) was measured on Powerscan4 (DS Pharma Biomedical, Osaka, Japan).

The cells were seeded at a density of 10⁵ cells/ml in 96-well plates and treated with 50-90 µM ZnSO₄ for 2 days as described previously^{38 (link)}. The alamarBlue reagent (AbD Serotec, Ltd., Oxford, UK) was added to the culture media and the cells were incubated for 4 h. Absorbance was determined at 570 and 600 nm using PowerScan 4 (DS Pharma Biomedical, Osaka, Japan).

The DF cells were seeded on a 96-well plate (5 × 10³ cells/well) for 12 h in DMEM containing 10% FBS. Thereafter, the medium was replaced with a medium containing 10% FBS with DMSO or 5 µM of each drug (dissolved in DMSO), and the plate was incubated for 24 h. The drugs used in the analysis are: auranofin, felodipine that was the second most effective drug after auranofin, and meloxicam, which has been used for DF in our institution as an NSAID^{4 (link)}. The activity of Caspase 3/7, which is an indicator of the apoptosis-inducing ability, was measured using a Caspase assay kit (Caspase-Glo 3/7 Assay, Promega) at the 24 h time point, and the luminous intensity was measured using a microplate reader, PowerScan4 (DS Pharma Biomedical, Osaka, Japan).
Similarly, in the ROS assay, 2.5 × 10⁴ T41A-mutated cells per well were seeded and incubated for 12 h, and then treated with a medium containing 10% FBS with DMSO or 1–10 µM of auranofin (dissolved in DMSO) for 24 h. The ROS Detection Assay Kit (BioVision, Waltham, MA) was used as described in its instructions, and fluorescence intensity was measured in the microplate reader.

DT40 cells deficient in ZnT1, ZnT4, and metallothionein (MT) genes (Δ1M4 cells) were generated previously.^{16 (link)} Wild-type DT40 cells and Δ1M4 cells were maintained in RPMI 1640 medium (Nacalai Tesque) supplemented with 10% heat inactivated fetal calf serum (Multiser, Trace Scientific Ltd.), 1% chicken serum (Invitrogen), and 50 μM 2-mercaptoethanol (Sigma-Aldrich) at 39.5°C, as described previously.^{16 (link)} Δ1M4 cells were stably transfected with plasmid containing C-terminally HA-tagged WT or mutant ZnT2 using electroporation.^{55 (link)} The transfected cells (10⁵ cells/mL) were seeded in 96-well plates and treated with 20–70 µM ZnSO₄ for 2 d. Alamar Blue reagent (AbD Serotec, Ltd.) was added to the culture media for 4 h, and absorbance was determined at 570 and 600 nm (PowerScan 4; DS Pharma Biomedical).

The PPIX concentration was measured as described previously [16 (link)]. First, 100 µL of cell suspension (5 × 10⁴ cells) were seeded into 96-well plates and cultured at 37 °C in the presence of 5% CO₂ for 24 h. The culture media was replaced with fresh media containing 0, 1.25, 2.5, or 5 mM 5-ALA and the cells were incubated at 37 °C in the presence of 5% CO₂. At 0 to 3 days post-treatment, the intracellular and extracellular PPIX concentrations were measured. The intracellular PPIX concentration was measured in cells treated with 100 µL of 1% sodium dodecyl sulfate after three times wash with PBS. The PPIX concentration in the cell supernatant was measured as the extracellular PPIX concentration. The fluorescence of each sample in a 100 µL was measured by POWERSCAN4 (DS Pharma Biomedical, Osaka, Japan) with emission wavelengths of 632 and 635 nm, after an excitation wavelength of 405 nm, to evaluate the intracellular and extracellular PPIX concentrations, respectively.