D luciferin

For LCI assays, the CDS of each target gene without the stop codon was cloned and transferred to target plasmids: pCAMBIA1300-NLuc for N-terminal luciferase fragment fusion or modified pCAMBIA1300-CLuc (CLuc is fused to the C-terminal of each gene) for C-terminal luciferase fragment fusion [40 (link)]. The plasmids were transformed into the GV3101 Agrobacterium strain. After collection by centrifugation, the Agrobacterium cells were resuspended in injection buffer (10 mM MgCl₂, 10 mM MES, pH 5.7, 200 μM Acetosyringone) and kept at room temperature for 4 h. Then, Agrobacterium cells with plasmid combinations and with P19 were mixed in a 1:1:1 ratio, and 50 μL of the mixture was injected into tobacco leaves. The injected tobacco plants were then incubated for 60 h. Finally, the tobacco leaves were sprayed with a D-luciferin (Biosynth, Staad, Switzerland) solution at a final concentration of 1 mM, and the luminescence intensity was observed after keeping the leaves in the dark for 7 min.

We purchased A2780 human ovarian cancer cells, U87-MG (#HTB-14), HEK293T (#CRL-3216), and MDA-MB-231 (#CRM-HTB-26) from ATCC (Manassas, VA, USA). LN308 cells (p53 null) were a kind gift from Markus Weiler (Heidelberg University, Germany). We purchased propidium iodide (#P4864), TBHQ (#112941), and doxorubicin (D1515) from Sigma-Aldrich (St. Louis, MO, USA), and DMSO (D159-4) from Fisher Scientific (Santa Clara, CA, USA). We used FBS (#26140087), Penicillin-Streptomycin-Glutamine (#10378016), and sodium bicarbonate (#25080094) from GIBCO BRL (Frederick, MD, USA). We used plasmid extraction kits and DNA gel elution kits from Qiagen (Valencia, CA, USA) and Epoch Life Sciences (Missouri City, TX, USA). We purchased D-Luciferin from Biosynth (Staad, Switzerland). Antibiotics for bacterial and cell culture experiments were from Sigma (St. Louis, MO, USA), and bacterial culture media was from Difco (Franklin Lakes, NJ, USA).

EAO cells were transiently transfected using FuGene HD (Promega) transfection reagent according to the manufacturer’s instructions. One day before transfection, 2 × 10⁴ cells per well were seeded in a white 96-well plate (Nunc by Thermo Fisher Scientific, Roskilde, Denmark) and incubated at 27 °C for 24 h. Each well was transfected independently with a 4:1 ratio of reagent:DNA with 100 ng plasmid DNA. The cells were transfected with the reporter construct zfPer1b-Luc^{29 (link)} or D-box_Cry1a-Luc based on^{31 (link)}. As a control, zebrafish PAC-2 cells^{30 (link)} were transiently transfected in the same 96-well plate. Non-transfected cells were used as negative control for each cell line. After incubation for 24 h, the transfection solution was replaced by culture medium supplemented with 2 mM D-Luciferin (Biosynth, Bratislava, Slovakia). Cells were exposed to four 12:12 h LD cycles followed by 24 h DD. The bioluminescence was measured with a Topcount NXT counter (PerkinElmer, Shelton, USA) as previously described^{29 (link)}. We imported the data into Excel (Microsoft) by using the "Import and Analysis" macro (S. Kay, Scripps Research Institute). Mean and standard deviation of eight independently transfected wells were calculated with R 3.6.1⁵⁹ and shown using ggplot2⁶⁰ .

Cytotoxicity of T-cell clones and transgenic T cells was analyzed by a bioluminescence-based lysis assay as recently described (21 (link)). In brief, Ma-Mel-86b and Ma-Mel-86c were engineered to express Firefly luciferase (Fluc) and cocultured with effector cells at an E:T ratio of 10:1 in the presence of 0.15 mg/mL D-Luciferin (Biosynth, St. Gallen, Switzerland). Subsequently, relative luminescence units (RLU) were determined over time in 3 h intervals using a FluoStar Omega plate reader (BMG Labtech, Ortenberg, Germany) and a 10 s integration time. Spontaneous cell death was measured in wells containing target cells only, maximum cell death was induced by exposure of target cells to Digitonin (Sigma) at a concentration of 30 µg/mL. Lysis was calculated using the following equation: lysis [%] = 100*[(spontaneous RLU—test unit RLU)/(spontaneous RLU—maximum RLU)].

Mice were anesthetized by i.p. injection of 80 mg/kg body weight ketamine hydrochloride (Pfizer) and 16 mg/kg xylazine (CP Pharma). Together with anesthetics, mice were injected with 150 mg/kg D-luciferin (Biosynth). After 10 min, BLI signals of the anesthetized mice were recorded using an IVIS Spectrum Imaging system (Caliper Life Sciences). For ex vivo imaging of internal organs 6 d after allo-HCT, mice were injected with D-luciferin and sacrificed 10 min later. Internal organs were removed and subjected to BLI. All pictures were taken with a maximum of 5-min exposure time and analyzed with the Living Image 4.0 software (Caliper Life Sciences) (14 (link), 19 (link)).