Lv200 bioluminescence imaging system

Wild-type HEK293T cells (2 × 10⁵) were seeded in a complete growth medium onto a 35 mm cell culture dish with a glass bottom (Greiner Bio-One, Kremsmünster, Austria). 20–24 h after plating the cells were transfected with a combination of plasmids bearing genes encoding the wild-type CST fused with either large (LgBiT) or small (SmBiT) subunit of NanoLuc luciferase (Table S5, 250 ng of each plasmid, 500 ng in total) using the FuGENE^® HD transfection reagent (Promega, Madison, WI, USA); 2–3 h before the measurement the conditioned medium was replaced with the serum-free OPTI-MEM medium (Life Technologies, Carlsbad, CA, USA). Immediately before the measurement, the Live Cell Reagent (Promega, Madison, WI, USA) was added to the cells according to a scaled-up protocol and the cells were immediately examined with an LV200 bioluminescence imaging system (Olympus, Tokyo, Japan).

5 × 10⁴ cells were grown in 400 µl growth medium containing 5% (v/v) BacMam-LgBiT on an 8-well chambered coverglass (Nunc Lab-Tek II) for 24 h. The medium was replaced with 100 µl of CO₂-Independent Medium containing Vivazine Live Cell Substrate (1:100 dilution) and incubated at 37 °C for 45 min before imaging. Images were acquired on the LV200 bioluminescence imaging system (Olympus) equipped with an ImagEM X2 EM-CCD camera (Hamamatsu) and a temperature-controlled stage. All images were captured with cellSens software (Olympus) with the indicated objectives, electron multiplying (EM) gain, and exposure settings (Table S8.) Further analysis and processing of images was performed using Fiji and was limited to pseudo-coloring and linear adjustments of contrast and brightness^{28 (link)}.

All imaging experiments were performed using the LV200 bioluminescence imaging system (Olympus) equipped with an ImagEM X2 EM-CCD camera (Hamamatsu) and a 60x, 1.4 NA objective. HEK293 cells transiently transfected with the indicated expression construct (1:10 dilution in carrier DNA) were plated onto Nunc Lab-Tek II 8-well chambered coverslips (Thermo Fisher) in 400 μl growth medium (DMEM + 10% FBS) at a density of 8Χ10⁵ cells per well. After 24 h of incubation at 37 °C, 100 μl of Nano-Glo Live Cell Reagent (Promega) was added. All images were acquired with cellSens software (Olympus) using electron multiplying (EM) gain of 600 and an exposure time of 3 seconds. Each image was generated using a average projection of 10 images. Generation of average projections, linear adjustments of dynamic range, and pseudocolor rendering were performed using Image J image processing software (Fiji package). Scale bar = 20 μm.

All imaging experiments were performed using the LV200 bioluminescence imaging system (Olympus) equipped with an ImagEM ×2 EM-CCD camera (Hamamatsu) and a ×40 oil, 1.4 NA objective. HEK293 cells and HeLa cells were transiently transfected as described above for the RAS cellular target engagement assays. Control plasmids encoding MAPK14 and HDAC–NanoLuc fusions were from Promega. Cells were plated onto Nunc Lab-Tek II eight-well chambered coverslips (ThermoFisher Scientific) coated with 0.1% gelatin in 200 µl of growth medium (DMEM + 10% FBS) at a density of 4 × 10⁵ cells per well. After 24 h of incubation at 37 °C, 100 µl of Nano-Glo Live Cell Reagent (Promega) was added. All images were acquired with cellSens software (Olympus) using an electron multiplying gain of 600 and an exposure time of 5 s. Each image was generated using an average projection of ten images. Generation of average projections and linear adjustments of dynamic range were performed using Image J image-processing software (Fiji package).

For real-time bioluminescence imaging analysis, age-matched pairs of mice (Fx/Fx control and BKO) were placed in DD for >30 days before harvesting the tissues. The tissue collection was repeated for a total of three pairs of mice. The tissues were processed and prepared in the same manner as in the luminometry recording, except that a glass bottom culture dish (MatTek, Ashland, MA) was used. The sealed culture was placed onto the stage chamber maintained at 36°C inside the LV200 Bioluminescence Imaging System (Olympus America, Irving, TX). The sample was imaged using a 10× objective lens with 10-min exposure time at 25-min interval for 7 days. The imaging files were converted to linear time-series by quantifying the signals on grid matrices. The heat map was generated using the top 200 time-series data starting with the strongest signals. The top 50 of these time-series data were analyzed for period, phase, and relative amplitude by FFT-NLLS as described above. The phases calculated by FFT-NLLS were further converted to the actual time and divided by each circadian period to normalize period differences. Statistical analysis was conducted between paired mice as described above.