Arvo mx 1420

Purified schizont stage parasites were adjusted to 10⁸ parasites/assay and firefly luciferase assays were performed using the ONE-Glo^TM Luciferase system (Promega) according to the manufacturer's instructions. NanoLuc luciferase activity was measured by the Nano-Glo® Luciferase Assay system (Promega) according to the manufacturer's instructions. Luciferase signals were immediately measured with a plate reader (ARVO MX 1420; PerkinElmer) for 15 sec. Plasmid copy numbers for each sample were estimated by targeting hDHFR with primers hDHFR.rtF and hDHFR.rtR as described above, and used to normalize the luciferase activities.

The TuMV strain that expresses yellow fluorescent protein (YFP)—TuMV-TuR1-YFP—and TuMV strain UK1 (Sánchez et al., 1998 (link)) were kindly provided by Dr T. Natsuaki (Utsunomiya University, Japan) and Dr F. Ponz (INIA, Spain), respectively. The spontaneous mutant UK1 m2 from UK1 virulent to Aki-masari carrying Rnt1-1 was obtained in our laboratory. These viruses had been maintained in the turnip cultivar Yukihime-kabu.
The second true leaves of B. rapa plants were dusted with carborundum and rub-inoculated with the inoculum 10 d after sowing. For Arabidopsis, four mature rosette leaves were inoculated 3 weeks after sowing in the same way as B. rapa. The extent of quantitative resistance to TuMV in plants was evaluated by counting the number of infection sites on the inoculated leaves using TuMV-TuR1-YFP. The infection sites were counted under blue light at 3–5 days post-inoculation (DPI). For the ao mutant of Arabidopsis, the regions infected by TuMV-TuR1-YFP on the non-inoculated upper leaves were harvested under blue light at 9 DPI, and viral levels were measured by ELISA using polyclonal antibodies against TuMV (Japan Plant Protection Association, Tokyo) according to the manufacturer’s instructions. Absorbance at 405nm was read using a microtiter plate reader ARVO MX 1420 (PerkinElmer Japan, Yokohama, Japan).

Cells were lysed in ice-cold buffer containing 25 mM Tris-HCl (pH 7.5), 0.2% Nonidet P-40, 1 mM dithiothreitol, 2 mM ATP, and 5 mM MgCl₂. The hydrolysis of the fluorogenic peptide, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-MCA) (Peptide Institute, Osaka, Japan) was measured in 50 mM Tris-HCl (pH 8.0) at 37°C by ARVO MX 1420 (PerkinElmer, Waltham, MA).

Luciferase expression vector under the control of the LXR-response element in the promoter (#336841, Qiagen) was transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen). After 48 h, the transfected cells were treated with 250 nM of Torin1 for indicated times. Luciferase activity was measured with the Dual-Glo Luciferase assay system (E2920, Promega, WI, USA) and an ARVO MX 1420 (Perkin-Elmer, MA, USA). Samples were measured in quintuplicate.

Cells (1.5 × 10⁴ cells/well) were seeded on a 96-well culture plate coated with 0.5 mg/mL of fibronectin in serum free medium. The relative number of viable cells was evaluated by the WST assay with a Cell Counting Kit-8 (DOJINDO) according to the manufacturer’s instructions. Absorbance at 450 nm was measured using a microplate reader (ARVO-MX 1420; PerkinElmer). The drug concentration producing 50% reduction in cell survival (IC₅₀) was calculated for each drug from linear regression analysis of the linear portion of the survival curves.