Cell-based Fluorescence Resonance Energy Transfer (FRET) assay

FRET occurs over short distances only (i.e., 2–10 nm) and, thus, represents a unique and powerful technique to monitor intermolecular protein interactions and target engagement in cells (Schaaf et al., 2017a, 2017b).

To develop an in-cell Skp1-F-box PPI assay, HEK293 cells were co-transfected with N-terminally mCherry-fused Skp1 (donor construct) and C-terminally copGFP-fused NIPA (acceptor construct) for 24 and 48 hrs (Figure S2B). To make sure that FRET is really happening, the cells were also transfected with a positive control (mCherry-copGFP fused) and a few negative controls (like unfused copGFP only, unfused mCherry, and unfused mCherry + copGFP). In addition, an empty vector transfection was used as a negative control to distinguish protein(s) expression in all systems. Microscopic immunofluorescence images revealed that protein expression in all systems was considerably enough at 48 hrs (Figure S2C). Next, we adopted the FRET system to 96-well format and, as shown in Figure S2C (bar graph), the average FRET fluorescence in our mCherry-Skp1 + NIPA-copGFP transfected system was comparable to that of the positive control. We further calculated the Z′ factor (as mentioned in the FP assay format) value from different batches of experiments (Z′ factor: 0.71), which suggested that our assay conditions were reliable to be adopted for compound(s) testing.

HEK293 cells at a density of 5 × 10⁴ cells/well in 100 μL of DMEM containing 10% FBS were seeded in white Greiner CellStar 96-well plates (Sigma). After 12 hrs of seeding, cells were co-transfected with 0.1 μg (100 ng) of the appropriate FRET constructs (mentioned in key resources table) (or an appropriate empty vector control) by Lipofectamine 2000 transfection according to the protocol of the manufacturer (LifeTech). The transfected cells were incubated at 37°C in a humidified incubator providing 5% CO₂, and media was replenished after 8–12 hrs of transfection. After 48 hrs of transfection, if there is compound testing, the chemical compound was added into cell growth media, without phenol red, in increasing concentrations (like 1 μM, 15 μM, and 30 μM) followed by incubation for 4 hrs under normal cell-growth conditions. Otherwise, the medium was removed and replaced with PBS (1 PBS washing). Plates were then analyzed on a Perkin Elmer instrument in fluorescence mode. For copGFP, excitation (Ex) was set at 482 nm and reading emission (Em) at 502 nm. For mCherry, Ex was set at 587 nm and reading Em at 610 nm. Background fluorescence was determined from wells transfected with pCDNA3.1 vector and subtracted. The FRET ratio (FR) was calculated by following equation.

FR = FRET_c ‒ FRET_b/FRET_GFPc; Where FRET_c and FRET_GFPc are fluorescence intensities of the cells, and FRET_b is the background fluorescence intensity of the wells containing media or PBS. The resulting data was analyzed in GraphPad Prism® v6.0 (San Diego, CA). The potential utility of this assay for investigating the inhibitor interventions against Skp1-F-box PPIs was affirmed by using the known Skp1 inhibitor 6-OAP as a positive control (Figure S2E). The FRET ratio was calculated as a measure of compound’s ability to inhibit Skp1-F-box protein complex formation.