Doxycycline hyclate doxy

Mouse iKras 4292 cell lines were obtained from Dr. Marina Pasca di Magliano [University of Michigan](Collins et al., 2012 (link)). Cells were maintained in RPMI 1640 media supplemented with 10% (v/v) FBS and 10 μg/ml doxycycline hyclate (doxy, Sigma Aldrich, Cat# D9891) for regular culture. For Kras^G12D induction experiments, cells were deprived of doxy at least 48 h for beginning experiments (0 h, baseline). Then, fresh media containing doxy 10 μg/ml was added, and cells maintained under culture during multiple time intervals (12, 24 and 48 h). Human hTERT-E6/E7-HPNE (ATCC Cat# CRL-4037, RRID: CVCL_C468) and hTERT-E6/E7-HPNE-KRAS^G12D (ATCC Cat# CRL-4039, RRID: CVCL_C470) pancreatic cell lines were obtained from ATCC and maintained in appropriate media according to recommendations. All cells were cultured at 37°C in a humidified incubator with 5% CO2 and were used to a maximum of 30 passages. All cell lines tested negative for Mycoplasma with last testing performed in July 2020.

Doxycycline: Doxycycline hyclate [Doxy] (Cat. D9891, Sigma-Aldrich) was prepared as a stock of 10 mg/mL solution for in vitro experiments and diluted to a concentration of 25 ng/mL in culture medium.
APR-246 (eprenetapopt): (Syngene, batch no: S0312053) was obtained from Aprea therapeutics (Solna, Sweden). APR-246 was prepared as a 100 mM stock in 100% DMSO (Cat. D8418, Merck) and stored in aliquots at −20 °C for in vitro and in vivo experiments. For in vitro experiments, stock solutions were diluted in complete media to reach the desired final concentrations of APR-246. Care was taken to keep the DMSO concentration consistent across different doses and between cell lines. APR-246 dose–response curves were determined using a range of concentrations (0–60 µM) and two biochemical assays (SRB and Alamar Blue assays). For in vivo experiments, the APR-246 stock solution (100 mM) was diluted to a 120 mg/mL working concentration using PBS. The working stock was aliquoted and stored at −20 °C. Before administering to the mice, the working stock was further diluted in PBS for IP injection as 200 mg/Kg/day per mouse for a period of 14 days.
XI-011: The MDM4 inhibitor XI-011 (Cat. AOB1263, AOBIOUS, Gloucester, MA, USA) was prepared in DMSO (Cat. D8418, Merck) at 4 mg/mL for in vitro studies (as previously described [27 (link)]).