A 1155463

For drug sensitivity assays, cells in the fertile culture were seeded on 12-well dishes at 18,000 cells/cm² and treated with selected drugs 24hrs later. The arid group was cultured for 0 (acute arid) or 14 (chronic arid) days under arid conditions prior to drug treatment. Cells supplemented daily for 4-5 days with fresh media supplemented with various drugs including: Gemcitabine (Pfizer), PHA-767491 (Cayman chemical), Silvestrol (Biovision), CPI-613 (Cayman chemical), UK 5099 (Cayman chemical) or A-1155463 (Cayman chemical) or vehicle (0.2% DMSO). Tumor cells were fixed at day 0 or endpoint (day 4-5) with paraformaldehyde 4% stained with 3 μM Hoecsht 33342 (Thermo) for 30’ at 37C and quantified using a fluorescence plate reader (Molecular devices) to assess cell numbers. Cell density was normalized for day 0 of each group.
For pharmacological inhibition of biosynthesis under serum repletion, treatment initiated with switch to arid conditions (acute arid) and cells were treated for 72hrs before readouts. Drugs used in that study include: Abemacicilib (MedChem Express), Rapamycin (ApexBio), Torin1 (MedChem Express) and Cycloheximide (Sigma).

PANC-1-GFP xenografts were established by injection of 8x10⁶ cells in DMEM into the flanks of male or female NOD.SCID mice. Tumors were allowed to grow to 7-9mm in diameter and then injected intratumorally using a 27g needle with 50μl with either PBS, A-1155463 (Cayman chemical, 1.25mg/ml) in buffer containing 10% DMSO, 40% PEG300, 5%l T80, 45% Saline, Buffer alone Gemcitabine (Pfizer, 1mg/ml) + albumin-conjugated paclitaxel (Abraxis, 0.6mg/ml) at a rate of ~50μl/minute. 24 hours post injection, Texas red-dextran 70 kDa was injected retro-orbitally 30 minutes prior to sacrifice. 4-6 sections per mouse (from different regions) were evaluated for apoptosis rates in GFP+ cells.