In vitro and in vivo BLI

Real-time BLI was performed using an In Vivo Imaging System (IVIS) Spectrum imager (PerkinElmer, USA) following the administration of furimazine—Nano-Glo Luciferase Assay substrate (Promega #N1120). For all in vitro BLI, cells were treated with 50 µM furimazine in Nano-Glo Luciferase Assay Buffer (Promega #N1120) for 5 min according to the manufacturer’s protocol. Then Bioluminescent images were captured with an open filter, binning set to 4. For in vivo BLI, animals were anesthetized with isoflurane prior to the subcutaneous injection of 250 µM furimazine (Promega #N1120; 1/20 dilution) into the calvaria defect site. Images were captured with an open filter, binning set to 4, and acquisition times of 60 s at the indicated settings. All BLI signal detected (both in vitro and in vivo) using the GpNLuc reporter represent BRET signal deriving from intramolecular energy transfer between NanoLuc and eGFP. Total flux (p/s) and average radiance (p/s/cm²/sr) were calculated using the Living Image software (PerkinElmer, USA).