Ms 8096w

The drug screening was performed in a 96-well plate format MS-8096W (Sumitomo Bakelite, Shinagawa, Tokyo, Japan). HuhT7-HAV/Luc cells were plated at a density of 2 × 10⁴ to 2.5 × 10⁴ cells/well. After 24 h, cells were treated with 10 µM of each of the 1134 drugs or DMSO alone. In the screening assay, DMSO was used as the control. After 24 h, luciferase activities were determined as HAV subgenomic replicon replication using luciferase reporter assays, and cell viabilities were determined with dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assays (Promega, Madison, WI, USA), as described previously [22 (link)].

A luminescent calcium assay using aequorin as an indicator was performed according to the previous reports (84 , 85 (link)). 293T cells were seeded into white-walled 96-well plates (MS-8096W, Sumitomo Bakelite) at 20,000 cells per well in Dulbecco's modified Eagle's medium/F-12 (FUJIFILM Wako) containing 10% FBS. After a day, cells were transfected with the expression plasmids of the opsin and the mitochondrial targeted aequorin N26D using polyethylenimine (PEI MAX, Polyscience). The ratio of polyethylenimine:plasmid was 4:1 in weight. The ratio of plasmids of opsin:aequorin (Figs. 2 and 3) and that of opsin:Gα:aequorin (Figs. 4 and 5) were 1:1 and 1:1:2 in weight, respectively. Six to eight hours after transfection, the medium was replaced with a fresh medium containing 2 μM 11CR or 5 μM ATR. The next day, the medium was replaced with an L-15 medium without phenol red (Invitrogen) containing 10% FBS and 10 μM coelenterazine h (FUJIFILM Wako) under dim red light. After 2 h of incubation in the dark, luminescence was measured using a microplate luminometer (Veritas, Turner Biosystems). The cells were stimulated with a handheld UV flashlight (0.11 ± 0.0036 mW mm⁻²; peak irradiance at 373 nm, Fig. S12) for 5 s.

The Caspase-Glo 3/7 assay (Promega, Madison, WI, USA) was used to determine caspase-3 and -7 activities according to the manufacturer's instructions. Briefly, 1×10⁴ cells were seeded onto 96-well white plates (MS-8096W, Sumitomo Bakelite, Tokyo, Japan). After 24 h, 0.5 µM thapsigargin was added and incubated for 6 h in 5% CO₂ at 37°C. Caspase-Glo 3/7 reagent was added at a 1∶1 ratio with the medium containing cells in each well of the 96-well plates, and left for 0.5 h at room temperature. Luminescence was recorded as a function of caspase-3 and -7 activities using the Luminescencer-JNR II AB-2300 (ATTO). Medium without cells was used as nonspecific background. The ratios of caspase-3 and -7 activities from each group relative to untreated control groups, defined as 1, were determined by luminescence.