Lb960

The CDS of TaVQ25-A was sub-cloned into the pGBKT7 vector. The plasmid was transformed into the AH109 yeast strain, and the empty pGBKT7 vector was used as a negative control. The yeast cells were first cultured on a selective medium (SD) without tryptophan (SD/-T), and the obtained positive yeast cells were then grown on SD plates without tryptophan, histidine, or adenine (SD/-T-L-H) and SD/-T-L-H plates containing X-α-D-galactosidase (X-α-gal) to observe their transcriptional activation activity.
The CDS of TaVQ25-A was sub-cloned into the pSAT-GAL4BD vector and transformed into wheat protoplasts. The transcription activation activity of TaVQ25-A was represented by the ratio of firefly luciferase (Luc), the reporter, to Renilla luciferase (Ren), the internal reference gene, and was measured using a microplate cold light detector (LB960, Berthold, Wildbad, Germany).

Cell viability was examined with the CellTiter-Glo^® luminescent cell viability assay kit (G7572, Promega). Cells (3 × 10³ cells/well) were seeded in 96-well plates with 200 μl of RPMI 1640 medium containing 10% FBS. Then, the cells were exposed to cisplatin at different concentrations for 48 h. Next, 25 μl CellTiter-Glo® reagent and 25 μl PBS were added per well on a vortex for 2 min. After incubating for 10 min to stabilize the luminescence signal, the plates were analyzed using a microplate luminous detector (LB960, Berthold, Germany).

LNCaP, HEK293-pcDNA3.1( +) and HEK293-GnRHR cells (1 × 10⁵ cells/well) were cultured in 48-well plates, then transfected with luciferase reporter plasmid containing cAMP response element (CRE) or androgen response element (ARE). In order to evaluate the transcriptional activity of YAP1, YAP1 knockout- and wildtype-LNCaP cells were cultured in 48-well plates and transfected with YAP/TAZ-responsive luciferase reporter plasmid (TEAD reporter). CRE-luciferase plasmid was purchased from Stratagene (La Jolla, CA, USA) and ARE-luciferase plasmid) [61 ] was kindly donated from Dr. Young Joo Lee (Department of Bioscience and Biotechnology, Sejong University, Republic of Korea. Lipofectamine 2000 (Thermo Fisher Scientific) was used per the manufacturer’s protocol. The transfected cells were exposed to compounds for 24 h, and the promoter activity was measured using a dual-luciferase reporter assay system (Promega, Madison, WI, USA). The firefly and hRenilla luciferase activities in the cell lysates were measured using a luminometer (LB960, Berthold Technologies, Bad Wildbad, Germany). The relative luciferase activities were calculated by normalizing the promoter-driven firefly luciferase activity to phRL-SV (hRenilla) luciferase (Promega, Madison, WI, USA).

Cellular ATP contents were measured by using an ATPlite assay kit according to the manufacturer's protocol (Perkin-Elmer Life Sciences). Briefly, 100 μl of the cell lysate were mixed with 100 μl of substrate solution and the plates were shaken for 2 min at 700 rpm using orbital plate shaker. The plates were then incubated for 10 min. Luminescence was measured by using a luminescence analyzer (Berthold LB960). Lactate was measured by using Lactate assay kit according to the manufacturer's protocol (Sigma-aldrich). Specifically, the cell culture supernatant was collected and deproteinized with a 10 kDa MWCO spin filter. The samples were then diluted using lactate assay buffer and mixed with reaction mixure (lactate probe, lactate enzyme mix), followed by incubation at room temperature for 30 min and subsequent measurement for absorbance at 570 nm.

The reporter plasmid and effector plasmid were constructed as described previously, with a little modification [48 (link)]. The reporter plasmid (CaMV 35S::REN-pSbMATE/pSbGlu1/pSbSTAR1/pSbSTAR2a/pSbSTAR2b-CaMV 35S (−46)::LUC) encodes two luciferases, of which the Renilla luciferase (REN) was controlled by the CaMV 35S promoter and the firefly luciferase (LUC) by the promoter indicated. CaMV 35S minimal promoter (−46) was synthesized [49 (link)] and inserted into the HindIII/BamHI sites of the vector pGreen-0800-LUC. The effector plasmid (CaMV 35S::Myc-SbWRKY22/SbWRKY65) was created by cloning the DNA fragment encoding SbWRKY22/SbWRKY65 into the vector pEGAD-Myc at the EcoRI site under the control of the CaMV 35S promoter. Primers are shown in Table S2. Arabidopsis protoplasts were isolated from 4-week-old plants. The protoplast/DNA mixture was incubated for 12–16 h in the dark at room temperature after the protoplasts were transformed using the PEG-mediated protoplast transformation method [23 (link)]. Finally, the transformed protoplasts were used for the dual-luciferase reporter assay according to the technical manual (Promega, E1910, Madison, WI, USA). The luminescent signal was measured with a luminometer (Berthold LB960, Bad Wildbad, Germany).

For the LUC bioluminescence assay, Col-0 and Pat were transformed with the p.PHYBpat::LUC and p.PHYBCol::LUC reporter using the floral dip method. Seedlings were grown directly on half-strength Murashige and Skoog 0.8% agar medium supplemented with 1% sucrose in a 96-well plate. One seed was placed per well and the seedlings were entrained under 16-h light/8-h dark cycles. After 7 days, 40 μl of luciferin (20 mM) was added to each well. The plate was transferred to constant light conditions and dark conditions and placed in a microplate luminometer LB-960 (Berthold Technologies, Bad Wildbad, Germany) to measure the bioluminescence emitted by each seedling every hour. Data analysis was conducted using the Mikrowin 2000 software (version 4.29, Labsis Laborsysteme GmbH, Neunkirchen-Seelscheid, Germany). Period estimates were calculated with Brass 3.0 software and analyzed using FFT-NLLS.