3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane

Western blot analysis was performed using GFP-specific antibody (Abcam Inc.). Protein was extracted from leaves of individual plant of N. benthamiana, A. thaliana, rose, and strawberry with 300 μl of extraction buffer [100mM TRIS, pH 6.8, 2.5% SDS, 100mM dithiothreitol (DTT), 100mM NaCl, 10% glycerol]. For Arabidopsis, protein was extracted from leaves of individual plants with 300 μl of extraction buffer (125mM TRIS-HCl, pH 6.8, 4% SDS, 20% glycerol, 2% β-mercaptoethanol). For chrysanthemum, protein was extracted from leaves of individual plants with 300 μl of extraction buffer [200mM HEPES, pH 8.0, 20mM EDTA, 100mM DTT, 200mM phenylmethylsulphonyl fluoride (PMSF), 0.2mg ml^–1 leupeptin, 50mg ml^–1 Polyclar VT]. Bradford assays were used to determine protein quantities, and equal amounts of proteins (10 μg) were separated by 10% SDS–PAGE. Proteins were transferred to a polyvinylidene difluoride membrane (GE Healthcare). CP–GFP was detected after an overnight incubation at room temperature with a 1:10 000 dilution of an anti-GFP antibody conjugated to alkaline phosphatase (Bedoya et al., 2010). Alkaline phosphatase was detected using a chemiluminescent substrate (CSPD; Roche) and exposed to X-ray film (Kodak X-OMAT BT Film/XBT-1).

Digoxigenin-labeled oligonucleotide probes were added to the prehybridization solution (50% formamide, 5 × SSC, 5% dextran sulfate, 0.2%SDS, 1 × Denhardt’s solution, 25 mM sodium phosphate pH 7.4, 100 μg/ml salmon sperm DNA). The nylon membrane of transfer printing was placed in the hybridization bag, and the prehybridization solution (20 ml/100 cm²) was added at 42 °C for 1 h. Hybridization solution was added and hybridized overnight at 42 °C. The next day, the hybrid solution was discarded, and the membrane was washed successively with solution A (2 × SSC, 0.1% SDS) for 5 min × 2 times, solution B (0.5 × SSC, 0.1% SDS) for 15 min × 2 times, and solution C (2 × SSC, 0.3% Tween20) for 1–5 min. Blocking buffer was added for 30 min, add anti-digoxin antibody (1:5000) was added for 30 min, the membrane was washed with solution C for 15 min × 2, the membrane was washed with CSPD diluent for 2–5 min, and CSPD working solution was added to the film for 5 min. The sealed nylon membrane was placed in a dark box at 37 °C for 10 min to enhance the luminescence. After exposure for a certain time, the membrane was removed for development and observation.

Cells were pre-cultivated in normal growth medium for four weeks and then either continued to be cultured in the same medium (reference) or switched to osteogenic medium. To measure osteogenic differentiation by alkaline phosphatase (ALP) activity, cell-seeded scaffolds were collected on days 0, 7, 14, and 21 after the point at which they were transferred to the osteogenic medium. The scaffolds were incubated for 1 h in serum-free medium, washed three times with PBS, and incubated in 2 mL lysis buffer for 20 min on ice. The lysis buffer consisted of PBS with 0.5% Triton^® X-100 (#30513, Carl Roth, Karlsruhe, Germany), 25 mM of TRIS (#10601, Carl Roth), 1 mM of MgCl₂ (#21892, Carl Roth), and 0.1 mM of ZnCl₂ (#Z015250G, Merck, Darmstadt, Germany). To ensure full lysis, the samples were subjected to three cycles of shock freezing (liquid nitrogen) and thawing and were then kept on ice. Special attention was paid to achieving lysis of all cells in the scaffolds. ALP activity was determined using an assay (0.4 mM CSPD™ substrate with Sapphire-II enhancer, #T2210, Thermo Fisher, Darmstadt, Germany) as a measure to determine the degree of cell differentiation to osteoblasts. A total of 20 µL of the lysate was mixed with 100 µL of the reagent in a non-transparent 96-well plate (triplicate measurement). After 20 min at room temperature (RT), chemiluminescence was measured for 0.5 s (Victor X4 multiplate reader, Perkin Elmer). Lysis buffer mixed with the ALP-reagent was used as negative control. A standard curve was generated for reference. Increased protein concentrations (0 to 200 µg/mL in 20 µg/mL steps) were generated from an albumin stock solution (fraction V, 1 mg/mL, #28341, Carl Roth) by dilution in lysis buffer (triplicate measurement). To normalize ALP activity to total protein content, protein concentration was determined using the Bradford assay (5× Roti^®Quant, #K015.2, Carl Roth) in a 96-well plate (triplicate measurement). After 5 min at RT, the absorption was measured (λ = 595 nm, 3 s).