Pierce firefly luciferase glow assay kit

COH2, H520, and SBC5 cells were seeded at a density of 3.0×10⁵ cells/well in 6 well plates and incubated overnight. PGL3-SOX2 promoter plasmid was purchased from Addgene (Cat #: 101761, Watertown, MA, USA). Transfection was performed with jetPrime (Polyplus, Cat #: 101000027, Graffenstaden, France) using 3 μg/well (COH2) and 4 μg/well (H520, SBC5) of plasmid DNA. After 12 h post transfection, the cells were harvested, counted, and seeded at a density of 2.0×10⁵ cells/well in a white 96-well assay plate (Corning, Cat #: 3917, New York, USA) and transparent 96-well plate. After cells adhered to the plate, ActD/CFZ/Cisplatin were added at IC50 concentration individually, or in combination and cultured for 12h/24h. The luciferase activity was measured using PierceTM Firefly luciferase Glow Assay Kit (ThermoFisher Scientific, Cat #: 16176), and cell viability was measured using CCK8. The luciferase activity and cell viability were correlated to determine the cages.

FTS was synthesized and purified following published literature.⁹ (link) PEI was purchased from Polysciences (Warrington, PA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and trypsin-EDTA solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pierce Firefly Luciferase Glow Assay Kit was purchased from Thermo Scientific (Waltham, MA, USA). OVA polyclonal antibody, fetal bovine serum (FBS), and penicillin-streptomycin solution were purchased from Invitrogen (Grand Island, NY, USA).

Wild type and rcm1Δsnr75Δsnr13Δ were transformed with pAC/PGK dual reporter vectors, consisting of a β-galactosidase gene, followed by a recoding window containing one of each TAA, TAG, and TGA stop codons (pAC/PGK-TAA/TAG/TGA), a −1 frame shift mutation (pAC/PGK-IBV), a +1 frameshift mutation (pAC/PGK-EST), or a control vector with no mutation (pAC/PGK-TQ), followed by the luciferase gene (Stahl et al. 1995 (link); Baudin-Baillieu et al. 2009 (link)). In total, 30 OD units of exponential cultures were lysed with glass beads in a lysis buffer (10 mM MgCl₂, 50 mM NaCl, 50 mM Tris at pH 7.4). To measure the β-galactosidase activity, half of the cell lysate was mixed with an equal volume of lysis buffer and 1 mM chlorophenolred-ß-D-galactopyranoside (CPRG, Roche), incubated at 37°C for 30 min and absorbance was measured at 575 nm on Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek). The remaining lysate was evaluated for luciferase activity, using the Pierce Firefly Luciferase Glow Assay Kit (Thermo Fischer Scientific) as per the manufacturer's instruction. The luminescence of samples was measured at 597 nm after 10 min incubation at room temperature.

Luciferase vectors containing wild-type and mutated binding sites of miR-143 in 3ʹUTR of IGF-IR were transfected into HUVECs using the Lipofectamine™ 3000 (Invitrogen, USA) according to the manufacturer’s instructions. After 24 hours’ transfection, relative luciferase activity was measured using the Pierce™ Firefly Luciferase Glow Assay Kit (Thermo Scientific, USA).