D luciferin solution

To assess the luciferase expression of the transduced BMSCs, different numbers of Luc-GFP-BMSCs (0.1, 0.2, 0.3, 0.4, and 0.5 × 10⁴/well) were seeded into a 96-well plate in 100 μL of growth medium, and D-luciferin solution (D-luciferin, 150 μg/mL, Gold Biotechnology, Inc., USA) was added at room temperature. After 10 min of incubation, the cells were imaged using an in vivo imaging system (IVIS) (PerkinElmer, IVISSPE, USA). The bioluminescent signals were analyzed using Living Image Software 4.5.

Graft‐bearing animals were injected IP with D‐luciferin solution (Goldbio) at 150 mg/kg 30 min before imaging to capture the peak in bioluminescent intensity. Mice were anesthetized with an isoflurane mixture (2% in 98% O₂), and the bioluminescent signal was quantified using a Xenogen IVIS 200 imaging system (PerkinElmer). Images were acquired for 1 min and then analyzed using the Living Image analysis software (Xenogen). Regions of interest (ROIs) were centered over the location of the devices, and background signal was obtained by capturing the ROI of a nonbioluminescent signal. Photons collected over the acquisition time were counted within the ROI. The same imaging protocol was applied each time to ensure consistency across longitudinal studies.

The full-length CDS of PINNA1, MtKNOX1, MtKNOX2, MtKNOX6, and MtKNOX7 were PCR amplified and fused to either the pCAMBIA3300-GFP or pCAMBIA3300-GR vector to generate effectors, respectively. The ∼2.9-kb promoter fragment upstream of MtUFO was cloned into the pGreenII-0800-Luc vector to generate a reporter construct. The constructs were introduced into A. tumefaciens GV3101 together with the pSoup19 helper plasmid. At least four tobacco leaves from independent plants were infiltrated and harvested after 48 h. For GR induction, another set of at least four tobacco leaves from independent plants were infiltrated with infiltration buffer containing 10 μM DEX (Coolaber, Cat: SL4070) or an equal volume of ethanol, and harvested after 48 h. The luciferase fluorescence signals were observed by a plant living imaging system (Tanon 5200, China) after these leaves were sprayed with 1 mM d-Luciferin solution (GoldBio, Cat: LUCK-1G) and incubated for 5 min in the dark. Then the rest of the leaves were punched and powdered in liquid nitrogen to measure the LUC and REN activity. Relative LUC and REN activity were measured by the DLR assay system on the Centro XS LB960 (Berthold, Germany). Finally, the relative firefly luciferase activity was calculated as the ratio of LUC to REN for each sample. The primer sequences are listed in Supplementary Data 1.

WS hMSCs implantation was performed as previously described (Zhang et al., 2015 (link)). In brief, 5 × 10⁵ luciferase-expressing WS hMSCs were pretreated with vehicle or 100 nmol/L Que for 30 days and then implanted into the middle of the tibialis anterior muscle of immunodeficient mice. Five days after the implantation, mice were anaesthetized and injected with D-luciferin solution (Gold-Bio, luck-500). After 15 min the in vivo luciferase activity of each mouse was determined by the IVIS lumina system (PerkinElmer). Luminescence intensity was normalized to luciferase intensity of hMSCs right before the implantation. All the animal experiments performed in this study were approved by the Institute of Biophysics, Chinese Academy of Sciences.