S5716

For drug response testing, organoids were harvested and diluted to 75 organoids/μL in the medium. 384-well plates were coated with 10 μL BME per well using a multidrop dispenser. 30 μL of the organoid suspension was then added to each well. Abemaciclib (1 μM final concentration, S5716, Selleck), PF-06873600 (0.4 μM final concentration, S8816, Selleck) and DMSO controls were then added in duplicate. After 7 days of drug treatment, cell viability was measured by adding 40 μL of CellTiter-Glo 3D Reagent (G9683, Promega) per well and reading luminescence after 5 min of shaking and 25 min of incubation in darkness at room temperature.

Cells were treated with 1 µM palbociclib (Cdk4/6 inhibitor, Selleckchem S1579), or with 0.5 µM abemaciclib (Cdk4/6 inhibitor, Selleckchem S5716) or with 1 µM K03861(Cdk2 inhibitor, Selleckchem S8100) for 16 h to synchronize cells at G1/S transition, and were collected (indicated by ‘G1 arrest’ in the figures). Alternatively, cells were then washed five times with PBS and released in S phase for 10 h before being collected. To extend G1 duration cells were treated with 160 nM palbociclib or with 50 nM abemaciclib or with 400 nM K03861 for 16 h and were collected (indicated by ‘G1 lengthening’ in the figures). Alternatively, cells were then washed 5 times in PBS and released in S phase for 10 h before being collected.
To inhibit DNA replication, cells were released in S phase in the presence of low doses of Aphidicolin (APH, A0781 from Sigma-Aldrich), a DNA replication polymerase inhibitor, or of PHA767491 (PZ0178 from Sigma-Aldrich), a Cdc7 inhibitor (indicated by ‘release in S phase + APH’ or ‘release in S phase + PHA’, respectively, in the figures). Doses were chosen to significantly decrease EdU incorporation without affecting the levels of DNA damage.