Nano glo live cell substrate

For live cell luminescence imaging, HEK-293 cells were plated on chambered coverslips coated with poly-L-lysine as described previously. After 24 h of incubation, the cells were cotransfected with the AR-FL and AR-V7 NanoBiT constructs (Suppl. Table 1) and incubated overnight. Immediately before imaging, the cells were stimulated with 1 nM DHT and NanoGlo live-cell substrate (Promega). Luminescence was recorded and integrated for a total of 90 min with an interval of 10 min and an exposure time of 10 s using a Leica DMi8 TIRF Widefield Fluorescence Microscope (Leica) equipped with an EMCCD camera (Andor, Oxford Instruments, iXon Ultra, Abingdon, UK).

β-arrestin recruitment was monitored using the NanoBiT PPI system (Promega). CCR7 and β-arrestin2 were C-terminally fused with LgBiT and N-terminally fused with SmBiT, respectively, and cloned into a pEF1α-IRES vector. HEK293 cells were transiently co-transfected in suspension with pEF1α-IRES CCR7-LgBiT(C) and pEF1α-IRES SmBiT-ARRβ2(N), as previously described. Transfected cells were seeded at a density of 1.5 × 10⁴ cells/well in white, clear flat-bottom 96-well plates coated with 100 µg/mL poly-D-lysin and incubated for 48 h at 37 °C and 5% CO₂. Next, cells were washed with an assay buffer (HBSS, 20 mM HEPES buffer (#15630-080, Thermo Fisher Scientific, Waltham, MA, USA), 0.5% FBS, pH 7.4) and incubated with 100 µL of a 1:100 Nano-Glo^® Live Cell Substrate (#N2012, Promega, Madison, MD, USA) working solution for 40 min at 37 °C and 5% CO₂. The plate was transferred to the FLIPR Tetra and baseline luminescence was measured for 30 s every 5 s. Thereafter, 25 µL of 5× ligand was added automatically to the cell plate by the FLIPR Tetra and changes in bioluminescence were monitored in real time for 40 min every 5 s. When required, 20 µL of 5× compound was added to the working solution Nano-Glo Live Cell Substrate. In the case of PTX, 25 µL of 5× concentrated compound was added after 24 h of incubation.

Split nanoluciferase constructs were generated by cloning full-length WT mB7-1, SER mB7-1, CYS mB7-1, WT mB7-2 and WT mPD-L1 into the NheI and XhoI of both the pBiT1.1-C (TK/LgBiT) and pBiT2.1-C (TK/SmBiT) vectors from Promega. For experiments, 1 mL of suspension HEK 293 cells were transfected with different combinations of SmBit and LgBit constructs (250 μg each) that were mixed and incubated with 2 μg PEI. Two days post transfection all transfected cell populations were counted twice and normalized to 1 x 10⁶ cells/ mL using Freestyle growth media (Invitrogen). For luminescence measurements, 50 μL of each normalized cell population were transferred to a 96-well half-well luminescence plate to which 12.5 μL of diluted Nano-Glo^® live cell substrate (Promega) was added. Cells were incubated with substrate for 10 minutes and then immediately read on an Envision plate reader (Perkin Elmer) to detect luminescence using a 5 sec integration time. Details regarding the competition experiments using the split nanoluciferase can be found in the supplemental information.
Additional methods are described in the Supplemental Methods.

All cell lines were obtained from the American Type Culture Collection (ATCC). Growth media, each supplemented with 10% FBS and 1% Penicillin/Streptomycin, for the cells were as follows: SKOV3 - DMEM, MDA-MB-468 – DMEM +1% MEM non-essential amino acids, MDA-MB-231 – RPMI 1640 + 0.1% Gentamicin, MDA-MB-453 - DMEM, SKBR3 – McCoy’s 5a Modified, and MCF7 – RPMI 1640 + 0.1% Gentamicin +4 mg/mL Insulin. Cells were maintained in 37 °C/5% CO₂. Recombinant HER2 ECD with C-terminal His tag was purchased from Speed Biosystems and reconstituted in PBS at 100 µg/mL. SHuffle T7 Express chemically competent E. coli cells were purchased from New England Biolabs. Cobalt Agarose Beads (High Density) and HTC Cartridges were purchased from Gold Biotechnology. Nano-Glo, Nano-Glo Live Cell Substrate (5 mM), and Cell-titer Glo 2.0 were purchased from Promega Corporation. Synthetic β9 and β10 peptides were purchased from Peptide 2.0. Protein purification was performed on an AKTA FPLC (GE Healthcare). Luminescence was measured on an Infinite M1000 Pro (Tecan).

S. pneumoniae cells were grown in C+Y medium at 37 °C until OD₅₉₅ = 0.2 and washed once with fresh C+Y medium. 1% NanoGlo Live Cell substrate (Promega) was then added, and luminescence was measured 15 times at 37 °C every 30 s in a plate reader (TECAN Infinite F200 Pro). Measurements were performed in triplicate and the average values were plotted, with the s.e.m. represented by the dot size.

For NanoBiT PPI assays, 3.0 × 10⁴ cells per well (RAS-less MEFs) were plated in a white-wall, clear-bottom 96-well plate (Thermo Scientific 165306) and incubated at 37 °C overnight. The next day, all wells were transfected with SmBiT-KRAS^G12V, and selected wells were transfected (technical replicates per experiment = 6) with either EGFR-LgBiT, LgBiT-KRAS^G12V, or LgBiT-KRAS^G12V α4-α5 mutants using PEI transfection method. Twenty four hours after transfection, media was aspirated from wells, and luminescence was measured using NanoGlo Live-Cell Substrate (Promega; Cat # N2012) suspended in Opti-MEM reduced serum media (Gibco; cat # 31985070). After the live-cell luminescence measurement, cells were lysed with 1.0% Triton X-100 and incubated with HiBiT peptide (0.1 μM) for 10 min on orbital shaker. Then, luminescence was measured to quantify LgBiT peptide levels. Live-cell luminescence was normalized to luminescence after HiBiT peptide addition, and all samples were normalized to SmBiT-KRAS^G12V/LgBiT-KRAS^G12V. Assays were performed three times each (n = 3).