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. 2016 Oct 4;6:34311. doi: 10.1038/srep34311

Figure 2. Construction of injectable BRET probe for specific detection of HIF activity.

Figure 2

(a) Light spectrum of POL bioluminescence and POL-AlexaFluor680 (POL-AF) BRET signals. (b) BRET generation by POL labeled with a NIRF dye AF680 (AF). Samples containing POL conjugated with AF (POL-AF), mixture of POL and AF (AF + POL), POL, or AF were observed in a 96-well plate for their light generation with indicated excitation (Ex) and emission (Em) filters. BL, bioluminescence; FL, fluorescence. (c) POL-AF stability in serum at 37 °C. POL-AF was incubated with 50% fetal calf serum (FCS) at 37 °C and BRET signal intensity was measured at indicated time by using IVIS-Spectrum with a filter of 700 nm. (d) Efficient delivery of POL-AF into cells through PTD function. LM8 cells were treated with POL-AF (POL) or OL-AF (OL), which lacks PTD sequences, for 30 min and then the harvested cells were examined their generation of BRET signals. The signals were calibrated based on the data shown in Fig. S3. The calibrated signal intensity from POL-treated cells was normalized by that of OL-treated cells. (e) Specific generation of BRET signals by POL-AF in hypoxic cells. Cancer cells were treated with POL-AF for 30 min under normoxic (21% O2) or hypoxic (1% O2) conditions and then BRET signals in the harvested cells were measured. (f) VHL-dependent regulation of BRET in 786-O cells. 786-O and VHL-reconstituted 786-O (VHL-786-O) cells are used in the assay. Cancer cells were treated with POL-AF for 30 min under normoxic (21% O2) conditions and then BRET signals in the cells were measured. (g) Oxygen-independent generation of BRET signal from POL with mutated ODD domain. LM8 cells treated with POL-AF or POmL-AF were incubated for 30 min under normoxic conditions and then BRET signals were measured. Relative BRET intensity in (e–g) was calculated by dividing signal intensities of the 1 h-cultured cells, which were incubated with fresh medium for 1 h followed by 30 min incubation with probes, by their corresponding signal intensity of the uncultured cells, which were trypsinized immediately after 30 min incubation with the probes (see Methods for details). n = 3, *p < 0.05. Error bars indicate s.e.m.