Orion 2 microplate luminometer

HEK293T cells were seeded in a 96-well plate at 50–60% confluence. After 24 h, cells were transfected with 120 ng of miR-29a mimics or a negative control. Cells were cotransfected with 30 ng of modified pGL3 plasmids containing the wild-type 3′-UTR of TET1, 2, and 3 or a mutant 3′-UTR of TET1, 2, and 3 with all putative target sequences mutated. Transfections were performed using 0.45 μl of Fugene (Promega, Madison, WI, USA). Cells were collected 48 h after transfection, and Renilla luciferase activity was measured using a dual-luciferase reporter system (Promega). Luciferase reporter assays were performed in duplicate and repeated in three independent experiments. Luciferase activity was detected using an Orion II microplate luminometer (Berthold Technologies, Bad Wildbad, Stuttgart, Germany).

B16-F10 cells were co-transfected with the NFκB-driven luciferase (Stratagene, La Jolla, CA, USA) vector and the Renilla reniformis luciferase reporter vector (Promega, Madison, WI, USA) at a ratio of 1:1/10 using Lipofectamine 3000 Reagent (Invitrogen; Carlsbad, CA, USA) for 4 h before being maintained with fresh medium for 24 h. Subsequently, cells were treated with MTII (10 nM) with or without lipopolysaccharide (LPS; 100 ng/mL) for 24 h. The NFκB-driven luciferase activities in cells were measured using a dual light kit (Promega, Madison, WI, USA) in Orion II microplate luminometer (Titertek Berthold; Pforzheim, Germany) and normalized with that of R. reniformis luciferase according to the manufacturer’s instructions.

Cells were seeded into a six-well plate (3 × 10⁵ cells/well) and transfected with 300 ng of pGL3-Control plasmid linearized by HindIII digestion (Promega, Madison, WI). One day after transfection, cells were treated with DMSO or indicated drugs for 24 h, and then subjected to the luciferase assay using a Luciferase Assay System (Promega, Madison, WI) according to the manufacturer’s instructions. Briefly, cells were rinsed with 1× PBS and then incubated with lysis reagent for 15 min on ice. Cells were scraped and the lysate was transferred to a microfuge tube. Cleared lysate after brief centrifugation to remove cell debris was transferred to a white 96-well microtiter plate and mixed with Luciferase Assay Reagent. Luminescence was measured by Orion II Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany) and normalized with the protein concentration to quantify the NHEJ efficiency. The HindIII restriction enzyme cuts the pGL3-Control plasmid between the SV40 promoter and the luc+ coding sequence. Luminescence can only be detected when the linearized plasmid is re-ligated by NHEJ and luciferase is expressed inside the cell. Luminescence in lysate of pGL3-Basic (with luc+ coding sequence but without an upstream promoter to drive luciferase expression) transfected cells was measured and served as the background signal.

NOTCH transcriptional activity was analyzed using luciferase assays, as previously described [18 (link),19 (link),22 (link),84 (link)]. We also treated C3H10T1/2 cells with the γ-secretase inhibitor DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) (10 μM), used as a NOTCH-signaling-inhibition control. Luciferase assays were measured using the Orion II microplate luminometer (Berthold), and samples were processed with the Dual-Luciferase Reporter Assay System (Promega), following the supplier’s recommendations. Assays were repeated at least three times.

For pseudovirus neutralisation experiments, Vero E6 were seeded in 96-well plates one day prior (6,000 cells/well). Heat-inactivated (56°C, 30 min) sera were serially titrated (4-fold titration series with 7 steps + buffer only control) in PBS, pseudovirus stocks added (1:1, v/v) and the mixtures incubated for 30 min at 37°C before being added to cells in triplicates (final on-cell dilution of sera: 20, 80, 320, 1,280, 5,120, 20,480, 81,920-fold). After an incubation period of 16-18 h, transduction efficiency was analysed. For this, the supernatant was removed, and cells were lysed by incubation with Cell Culture Lysis Reagent (Promega) at room temperature. Lysates were then transferred into white 96-well plates and luciferase activity was measured using a commercially available substrate (Luciferase Assay System, Promega) and a plate luminometer (Orion II Microplate Luminometer, Berthold). For analysis of raw values (RLU/s), background signal of an uninfected plate was subtracted and values normalized to pseudovirus treated with PBS only. Results are given as serum dilution resulting in 50% virus neutralization (NT50) on cells, calculated by nonlinear regression ([Inhibitor] vs. normalized response – Variable slope) in GraphPad Prism Version 9.1.1.