One glo tox luciferase reporter and cell viability assay

First, the p62-fluc.Npc1^+/+ and p62-fluc.Npc1^−/− MEFs were plated into white 96 well plates (2 k cell/well with 100 μL media) and allowed to settle for 24 hours. Next, the MEFs were treated with 1 μg/ml doxycycline (Sigma-Aldrich 33429) for 24 hours before being washed off 3 times with PBS. Subsequently, the specified drug or a combination of drugs was dissolved in Opti-MEM (Thermo Fisher) and all of media was removed from cells and replaced with DSPE-PEG_2k containing media and incubated for 24 hours. MEFs to be treated for 48 hours with the micelles were aspirated after 24 hours and fresh DSPE-PEG_2k media was added for the final 24 hours. After 24 hours or 48 hours of drug treatment, MEFs were analyzed using ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay (Promega) by following the manufacturer’s protocol.

To perform the automated drug screening, 30–40,000 cells were grown in white multiwell plates, added with drugs, and analyzed 24 h later. As the screening of the PHOX2B 3′UTR was performed in this study for the first time, no positive or negative control was known. Therefore, while the PHOX2B promoter screening (promoter HTS) was conducted in duplicate and in the presence of positive/negative controls, to obtain the most reliable screening even in the absence of controls, the 3′UTR screening (3UTR HTS) was carried out in triplicate. Both the luminescence produced by the firefly luciferase gene and the fluorescence produced by protease-mediated activity on the glycylphenylalanyl-aminofluorocoumarin (GF-AFC) substrate in living cells were measured by using a two-step assay which yielded the double information in a single well (ONE-Glo^™ + Tox Luciferase Reporter and Cell Viability Assay, Promega). Either parameters were measured by an automated microplate reader (GloMax^® Promega) following an already set up procedure [14 (link)]. To avoid misleading results that might be ascribed to the effect of drugs on the regulatory regions upstream or downstream the renilla gene, in the 3UTR HTS, renilla firefly activity was not measured.