Glomax navigator system

IC₅₀ is the drug concentration at which half of the cells undergo cell death; it serves as an indicator of cellular toxicity. Cell viability and IC₅₀ values of various vasodilators (lidocaine, papaverine, nitroglycerin, phentolamine, and olprinone) were investigated using RSMCs and HCAECs. IC₅₀ values were measured using the GloMax^® Navigator System (Promega, Madison, WI, USA). The test compounds were diluted in the culture medium and stored in an incubator until use. The concentration of each compound was determined based on its potency to induce maximum vasodilation in the rat abdominal aorta 10 min after administration, as measured using the wire myograph system described earlier. For each test compound, a control and 11 different concentrations were prepared, and the cytotoxicity after the addition of the compounds was examined. The concentration of each compound is listed in Table 1. The RealTime-GloTM MT Cell Viability Assay kit (Promega, cat#G9711, USA) was used, and the substrate and Nano-luc were added to the medium in each well of a 96-well plate seeded with RSMCs and HCAECs, with 50 μL per well. The plate was incubated for 1–3 h in the incubator. Then, 50 μL of each prepared test compound was added to the plate. The IC₅₀ values were measured using GloMax at 1, 5, 10, 30, and 60 min after the addition of the test compounds, and cytotoxicity was evaluated.

An ATP-based cell viability assay was performed to measure the number of metabolically active hGF-1 and hOK (CellTiterGlo 2.0 Assay, Promega, Madison, WI, USA) after exposure to the eluates. The assay reagent was prepared according to the manufacturer’s recommendations and added to the cell suspension to lyse the cells and release their intracellular ATP. Upon exposure to ATP, the assay returned a luminescence signal (relative light units, RLU) which was measured in a luminometer (GloMax Navigator System, Promega). Cell viability was computed as the percentage of the control group (cell culture medium containing no agent).

In our reported gene assays, pNL1.2 (#N1001, Promega) was used as a vector. Fragment DNA was inserted into the pNL1.2 vector using Ligation high ver.2 (LGK-201, Toyobo) with KpnI (#R0142S; New England Biolabs, Inc.) and NheI (#R0131S; New England Biolabs) according to the manufacturer's protocol. Transformation was performed using Competent Quick DH5a (DNA-913F; Toyobo), and plasmids were purified using NucleoBond Xtra Maxi (Macherey-Nagel) according to the manufacturer's instructions. Transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol. Cells were assayed after vorinostat or vehicle treatment for 6 hours using the Nano-Glo Luciferase Assay System (N1120; Promega) with the GloMax Navigator System (Promega) according to the manufacturer's instructions. A measurement time of 1 second was used for NanoDLR, and the relative promoter activity was calculated.

Approximately 1 × 10⁵ HEK293 cells were seeded in flat-bottom 24-well plates (Thermo Fisher Scientific) and incubated overnight. The following day, the cells were cotransfected with the Cas13 variant-expressing plasmid (pCAGEN series, 150 ng), the gRNA-expressing plasmid (pC016 series, 300 ng), and the HiBiT reporter plasmid [pAG3-(GGGGCC)₆₆-HiBiT-GR frame and GP frame, 400 ng; pAG3-(GGGGCC)₆₆-HiBiT-GA frame, 75 ng] using TransIT-293 (Mirus) according to the manufacturer’s instructions. Forty-eight hours posttransfection, 400 μl of the medium was removed from each well, and 100 μl of HiBiT Lytic Reagent (Nano Glo HiBiT Lytic Detection System, Promega) was added to the remaining 100 μl of the medium. After incubating for 10 min on an orbital shaker, the total lysate was transferred to a 96-well flat-bottom white microplate (Greiner Bio-One or Corning), and luminescence was measured by the GloMax Navigator System (Promega).

GAL4-luciferase reporter assays were performed as previously described, with minor modifications 38 (link). Briefly, HEK293T cells were transfected with a pGL3 firefly reporter plasmid containing five tandem GAL4 DNA binding sites (GAL4-luc) and a pM-GAL4-YAP1 (TA-domain) effector plasmid. Luciferase assays were performed with the GloMax® Navigator System (Promega). To test the transcriptional activity of YAP1 on its downstream target genes, HEK293T cells were transfected with pGL4.1 firefly reporter plasmid carrying CTGF or CYR61 and the YAP1 isoform-expressing plasmids. The results were normalized to renilla luciferase activity and are expressed as the mean fold induction. Mean values of at least three independent experiments are displayed as the mean±S.D.