Glomax 96 microplate luminometer

Reactive oxygen species (ROS) production was monitored with an L-012/peroxidase-based assay on leaf discs collected from N. benthamiana, soybean, tomato, and Arabidopsis plants. The leaf discs were floated overnight on 200 μL of ddH₂O in a 96-well plate. The ddH₂O was replaced with a working solution [20 μM L-012 (Waco), 20 μg/mL peroxidase (Sigma-Aldrich), 1 μM flg22 (Sangon), or 1 μM purified protein reaction solution]. After the addition of the working solution, the plate was immediately moved to a GLOMAX96 microplate luminometer (Promega, Madison, WI, USA) for measurement of luminescence.
ROS detection in soybean leaves in response to zoospores of different P. sojae strains was monitored with an L-012 /peroxidase-based assays as previous reported with some minor modifications^{63 (link)}. Briefly, Leaf discs was collected from 2-week-old soybean plants and then floated overnight on 200 μL of ddH₂O in a 96-well plate. The ddH₂O was replaced with a working solution [20 μM L-012 (Waco), 20 μg/mL peroxidase (Sigma-Aldrich), zoospores (10000 zoospores per mL) of WT, PsRLK6-knockout (ΔPsRLK6) or PsRLK6-GFP overexpression transformants (OT-24). After the addition of the working solution, the plate was immediately moved to a GLOMAX96 microplate luminometer (Promega, Madison, WI, USA) for measurement of luminescence.

HEK-293T cells were purchased from Beijing Beina Chuanglian Biotechnology Institute (China), and 10 to 20 passages of cells were used in this experiment. Cells were seeded at densities of 1 × 10⁵/well into 24-well plates and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (SH30243.01, Hyclone, Logan, UT, USA) supplemented with 100 IU/mL streptomycin, 100 IU/mL penicillin, and 10% fetal bovine serum (FBS) (A31608-02, Gibco, Carlsbad, CA, USA). When cells reached a confluence of 85 to 90%, lipofectamine 2000 (11668019, Life Technologies Inc., Waltham, MA, USA) was used to cotransfect the cells with the miR-181 mimics (100 nM), pmirGLO-ZIP14 3′UTR-wt (50 ng) or pmirGLO-ZIP14 3′UTR-mut (50 ng). At 48 h post-transfection, luciferase activity was detected with a GlOMAXTM 96 microplate luminometer (E1910, Promega, Madison, WI, USA). Syntheses of mimics of miR-181 family members (miR-181a-5p, miR-181b-5p, miR-181c-5p, and miR-181d-5p) were performed by Biomics Biotechnologies Co., Ltd. (Nantong, Jiangsu, China). These sequences are shown in Table S6.

The AR antagonist effect of tested compounds was evaluated using the luciferase reporter gene assay (Xu et al., 2014 (link); Xu et al., 2015 (link); Zuo et al., 2017 (link); Xu et al., 2018 (link)). Briefly, RLUs were used to indicate firefly and Renilla luciferase activity, and the activities were evaluated using dual luciferase assay kits (Promega) in accordance with the manufacturer’s instructions. RLUs were measured using a GloMaxTM 96-Microplate Luminometer (Promega) and three individual experiments were performed as the mean ± SEM. For agonists, fold of induction = LU_induced/RLU_uninduced. For antagonists, % of control = 100 × RLU (agonist + antagonist)/RLU (agonist alone). All RLUs were normalized according to firefly RLUs/Renilla RLUs. EC₅₀/IC₅₀ values were expressed as μM, and Graph-pad Prism 5 software was used to calculate the IC₅₀ of phenylephrine (μM) by plotting the data using nonlinear regression analysis.