Nanoluc fusion vector

The BTK(C481S)-NanoLuc^® Fusion Vector was purchased from Promega, and siBTK was purchased from GE-Healthcare Bio-sciences (Accell Human BTK siRNA, EQ-003107–00–0005, 29121299, sequence supplied in Table S3). MM1R cells were transfected with siBTK alone or siBTK combined with BTK(C481S) using the Nucleofector IIb device (Lonza, Switzerland) according to the manufacturer’s instructions. In short, 2.10⁶ MM1R cells were resuspended in supplemented nucleofector solution, after which 300 nM siBTK was added with or without 2 µg BTK(C481S). To each nucleofection reaction, an additional 2 µg of pmaxGFP™ Vector was added as an internal positive control (transfection efficiency = 51.3 ± 2.4%). Resuspended cells were subsequently transferred to a cuvette and transfected using the O-020 nucleofector program. After nucleofection, a pre-equilibrated medium was added, and cells were transferred to a 96-well plate at a density of 80.000 cells/well. Eight hours post-transfection, transfection efficiency and GFP expression were assessed with fluorescence microscopy. If a GFP signal was present, cells were treated with increasing concentrations of WA and cell viability was measured after 24 h using the MTT colorimetric method. BTK mRNA expression of nucleofected cells was further confirmed by quantitative real-time PCR.

To determine the affinity of RP-6306 in the PKMYT1 or WEE1 NanoBRET target engagement assay (Promega), HEK293T cells were transfected with a NanoLuc fusion vector for PKMYT1 (Promega NV1871) or WEE1 (Promega NV2231) with transfection carrier DNA (Promega E4881) using Fugene HD Transfection reagent (Promega E2311) in Opti-MEM without phenol red (Thermo Fisher Scientific, 11058021) and incubated overnight. Cells were trypsinized, counted and 17,000 cells per well were plated in 96-well plates with K-5 cell-permeable kinase NanoBRET TE tracer (Promega N2482) and RP-6306 and incubated for 2 h at 37 °C. Intracellular TE Nano-Glo Substrate/Inhibitor (Promega N2160) was added, and the intensity of the acceptor emission (610 nm) and the donor emission (450 nm) were measured using an EnVison plate reader (Perkin-Elmer).