Special cell culture assays

2.2.7.1 Density dependence experiment Bioluminescence imaging

U-2 OS cells harboring a Per2:Luc or Bmal1:Luc reporter gene were harvested as described. For each seeding density, concentration of the cell suspension was adjusted to allow for seeding of the indicated cell number in equal seeding volumes, i.e. 1 mL/35-mm dish or 1 mL/membrane insert. Membrane inserts were placed in empty 35-mm dishes. All dishes were supplemented with 2 mL/dish complete culture medium and incubated under standard tissue culture conditions overnight. Following incubation cells were prepared for bioluminescence imaging and luciferase activity was continuously monitored.

RT-qPCR, RNA sequencing

U-2 OS cells were seeded at dense (3.0 x10⁵ cells/well) or sparse (0.3 x10⁵ cells/well) culture density into 6-well dishes. Wells were supplemented with 2 mL/well complete culture medium and cells were incubated under standard tissue culture conditions overnight. Following incubation, cells were synchronized and supplemented with 2 mL/well serum-free culture medium. Cells were incubated for 18 hours under standard tissue culture conditions. Following incubation, RNA was isolated and RT-qPCR or RNA sequencing performed as described.

2.2.7.2 Phase-pulling experiments

U-2 OS cells harboring a Per2:Luc reporter gene and U-2 OS non-reporter cells were passaged into two T-75 cm² flasks each. Upon confluence cells were separated into

“early” and “late” cells (one flask per cell line and timepoint), which were prepared for co-culture experiments 6 hours apart. At both timepoints, U-2 OS reporter and non-reporter cells were harvested and counted as described. Synchronization was

performed in solution by adding 1 µM Dexamethasone and incubating in a 37°C water bath for 30 minutes. For each co-culture ratio, concentrations of the cell suspensions were adjusted in serum-free culture medium to allow for seeding of the indicated cell numbers in equal seeding volumes, i.e. 100 µL/35-mm dish as indicated below (Table 2-2). “Early” cells were supplemented with 2 mL/dish reporter medium and incubated for 6 hours under standard tissue culture conditions. “Late” cells were prepared 6 hours later by the same procedure and were carefully added to 35-mm dishes containing the

“early” cells, i.e. 100 µL/dish and as indicated below (Table 2-2). This way low-density U-2 OS Per2:Luc reporter cell were co-seeded with increasing numbers of 6 hour phase advanced or phase delayed non-reporter cells, while keeping the absolute culture density constant (total of 3.3 x10⁵ cells/dish at seeding). 35-mm dishes were transferred to the LumiCycle and luciferase activity was continuously monitored.

Table 2-2: Seeding numbers of phase-pulling experiments Early

*6 hour phase delayed non-reporter cells relative to co-cultured early U-2 OS Per2:Luc reporter and non-reporter cells

**6 hour phase advanced non-reporter cells relative to co-cultured late U-2 OS Per2:Luc reporter and non-reporter cells

2.2.7.3 Period-pulling experiments

U-2 OS wildtype, CRY2^-/-, and TNPO1^-/- knock-out cells harboring a circadian Bmal1:Luc reporter gene, as well as U-2 OS wildtype non-reporter cells were harvested as described. Concentrations of the cell suspensions were adjusted in complete culture medium to 3.0 x10⁶ cells/mL. Mixed suspensions were prepared by mixing Bmal1:Luc reporter (either wildtype, CRY2^-/- or TNPO1^-/- knock-out) and non-reporter (wildtype) cells in a 1:5 ratio. Subsequently, 20-30 10 µL droplets (3.0 x10³

cells/droplet) of pure reporter or mixed reporter:non-reporter cell suspensions were seeded on inverted lids of sterile 100-mm petri dishes. Dishes were supplemented with 10 mL/dish 1X PBS to prevent evaporation of droplets and lids were placed on the dish. Hanging drops were incubated under standard tissue culture conditions for 5-7 days. This way spheroids were generated by gravity dependent cell aggregate formation and 3-dimensional outgrowth. Spheroids were harvested by pipetting and transferred to a 15 mL sterile tube. Remaining supernatant was aspirated and spheroids were re-suspended in 50-100 µL Matrigel for seeding in pre-warmed 35-mm dishes. Basement matrix was solidified at 37°C for 1 hour and spheroids were supplemented 2mL/dish complete culture medium. Spheroids were incubated under standard tissue culture conditions overnight. Following incubation spheroids were synchronized and prepared for bioluminescence imaging as described. Luciferase activity was continuously monitored.

2.2.7.4 Amplitude and damping rescue experiments

U-2 OS cells harboring a Bmal1:Luc reporter gene and U-2 OS non-reporter cells were harvested as described. Sparse reporter cells (0.25 x10⁵ cells/dish or insert) were seeded into 35-mm dishes or on 4.2 cm² membrane inserts. Membrane inserts were placed into empty 35-mm dishes. For each seeding density of non-reporter cells, concentration of the cell suspensions was adjusted to allow for seeding of the indicated cell number in equal seeding volumes, i.e.1 mL/35-mm dish. For direct co-cultures non-reporter cells were directly added to dishes containing non-reporter cells. For membrane separated co-cultures non-reporter cells were seeded into separate 35-mm dishes.

Subsequently, all dishes were supplemented with 2 mL/dish complete culture medium and incubated under standard tissue culture conditions overnight. Following incubation cells were synchronized and prepared for bioluminescence imaging as described.

Membrane inserts with reporter cells were transferred to dishes containing non-reporter cells. Ultimately, luciferase activity was continuously monitored.

2.2.7.5 Temperature pulse experiments

U-2 OS cells harboring a Bmal1:Luc reporter gene were seeded at dense (3.0x10⁵ cells/dish) or sparse culture density (0.4 x10⁵ cells/dish) into 35-mm dishes. Cells were

supplemented with 2 mL/dish complete culture medium containing 1:200 LY2109761 or DMSO (5 µM and 0.5% final concentration respectively) and incubated under standard tissue culture conditions overnight. Following incubation, cells were synchronized and supplemented with 2.5 mL/dish reporter medium containing 1:200 LY2109761 or DMSO (5 µM and 0.5% final concentration respectively). Temperature pulse experiments were performed in the LumiBoxen using Jumo Imago software (Table 2-3) in order to apply an 8 hour, 20°C temperature pulse at the inferred trough of PER2 expression (anti-phasic to Bmal1:Luc expression).

Table 2-3: Temperature pulse protocol (Jumo Imago)

Density Reagent Pulse No pulse control

Dense DMSO 64 hours 37°C // 8 hours 20°C // hold 37°C

hold 37°C

Dense LY2109761 70 hours 37°C // 8 hours 20°C // hold 37°C

hold 37°C

Sparse DMSO 67 hours 37°C // 8 hours 20°C // hold 37°C

hold 37°C

Sparse LY2109761 73 hours 37°C // 8 hours 20°C // hold 37°C

hold 37°C