Nano glo live cell assay system

One day before transfection, HEK293T cells were seeded at a density of 2 × 10⁴ cells/well in a 96‐well plate containing MEM‐α with charcoal‐stripped FBS. On the following day, expression vectors and PEI were diluted in 16 μL of Opti‐MEM (Gibco). Of note, 6.25 ng·μL⁻¹ of LgBiT‐AR and AR‐SmBiT or LgBiT‐AR P767A and AR P767A‐SmBiT expression vectors were used for the AR dimer formation assay. Further, AR‐LgBiT and FKBP‐SmBiT; LgBiT‐HSP90 and SmBiT‐FKBP52; AR‐LgBiT; and LgBiT‐FKBP51, AR‐LgBiT, and SmBiT‐HSP90 expression vectors were used for the protein–protein interaction assays. Diluted vectors and PEI were combined and vortexed and then incubated for 30 min at room temperature. After incubation, the solution mixture was directly added to the cells drop‐wise. Two days after transfection, the luminescence was measured using a Nano‐Glo^® Live Cell Assay System (Promega) and Nivo (PerkinElmer, Waltham, MA, USA) or ARVO X4 (PerkinElmer) or GloMax^® Navigator (Promega). DHT or MeOH was added 4 h before or 15 min after the addition of the luminescence reagent.

To assess the influence of antivirals regarding their specific activity during nuclear egress, the HCMV core NEC NanoBiT interaction system was applied. 293T cells (5 × 10⁵ cells per 6-well) were transfected with 2 µg of corresponding expression constructs (cotransfection of LgBit and SmBit constructs, or single transfection with an empty vector as background control) using PEI, as described in 2.1. At 1 to 2 d post transfection (d p.t.), biological triplicates of cells were transferred in equal confluences into a 96-well plate (white plate with clear bottom), and 3 to 4 h later, the antivirals, diluted in standard cell culture medium, were added in suitable concentrations. Directly after compound supplementation, a luciferase assay was performed using the NanoGlo Live Cell Assay System, according to the manufacturer’s protocol (N2011, Promega, Madison, WI, USA). The luciferase activity was measured at 37 °C for 1.5 h using the Victor X4 microplate reader. Background activity of LgBit luciferase activity without the SmBit counterpart was subtracted from LgBit/SmBit cotransfected cells to obtain PPI-specific luciferase activity.

Parental or DPH2KD HeLa cells were seeded in 24 well plates before being co-transfected with plasmids encoding NLuc-Hsp70 and plasmids encoding either mock, or DTA-HA, or Palm-DTA-HA. 6 h after transfection, cells were detached and split in triplicate wells of a 96 well plate. 24 h later, cells were washed with DPBS and NanoLuc activity was measured in each well using the Nano-Glo Live Cell Assay System (Promega, Wisconsin, USA) following the manufacturer’s instructions using the iD3 SpectraMax microplate reader (Molecular Devices, California, USA). The percentage of protein synthesis was calculated relative to the mock-transfected cells (mock set at 100%) for each cell type tested.

We used Nano-Glo^® Live Cell Assay System (Promega) for the split luciferase reconstruction assay. HEK293T cells were plated on poly-d-lysine-coated 96-well plates. Plasmid DNAs were transfected using Viafect. Each protein was sub-cloned into pBiT1.1-C [TK/LgBiT], pBiT2.1-C [TK/SmBiT], pBiT1.1-N [TK/LgBiT], and pBiT2.1-N [TK/SmBiT] vector. Plasmids were transfected into HEK293T cells in different combinations. At 24 h post-transfection, the medium was replaced with 50 µL of D-MEM and we followed the manufacturer’s protocols for measuring luciferase activities. Luminescence was measured using a GloMax^®-Multi Detection System (Promega).

EVs were obtained from parent or DPH2KD HeLa cells transfected with a plasmid encoding NLuc-Hsp70. Acceptor parent HeLa cells were seeded in 96 well plates the day before EV incubation. The same protein amount of EVs for each condition is incubated with acceptor cells for 24 h. After incubation, acceptor cells are washed twice in DPBS and NanoLuc activity was measured in each well using the Nano-Glo Live Cell Assay System (Promega, Wisconsin, USA) following the manufacturer’s instructions using the iD3 SpectraMax microplate reader (Molecular Devices, California, USA). EV uptake is shown as percent of input material. Statistical significance is obtained through an unpaired t-test.

Fusion constructs of mouse Rbm15 and Wtap to NanoBiT subunits were generated as follows: Full-length Rbm15- and Wtap-coding sequences were amplified with the oligonucleotides indicated in Supplemental Table 2 from poly-A-selected mRNA using NEBNext High-Fidelity 2X PCR master mix (New England BioLabs). Overhangs with homology to destination vectors (pBiT1.1-C, pBiT2.1-C, pBiT1.1-N, and pBiTN.1-C; Promega) were included in oligonucleotide sequences. Gel-purified PCR products were cloned into EcoRI sites using NEBuilder HiFi DNA assembly kit (New England BioLabs) following the manufacturer's recommendations. The optimal combinations of N-terminal- or C-terminal-tagged fusions to small or large subunits were determined through transfection of 20,000 wild-type mESCs per well with Lipofectamine 3000 reagent (Invitrogen) seeded in 96-well tissue culture plates (Corning, catalog no. 3917). Measurements were performed using the Nano-Glo live-cell assay system (Promega) and measured in a microplate luminometer (Berthold, LB960). The Rbm15/Wtap fusion combination yielding the highest luciferase activity was then transfected into distinct mESC genetic backgrounds, and the expression level of the fusion construct was quantified via RT-qPCR using oligonucleotides described in Supplemental Table 2.

To assess BMP2 growth factor activity, TC28a2-BRE-PEST cells (previously described^{96 (link)}) were stimulated with BMP2 at a range of concentrations (0.1–1000 ng/ml). Nano-Glo® live cell assay system (Promega, #N2011) was used for analysis of luminescence after stimulation at different time points. Luminescence was measured in raw luminescent units (RLU) using a GloMax® multimodal plate reader (Promega). Reads across a time course were performed using separate wells.