Pd0325901

Bovine FFs were obtained from 40 to 45 day-old male fetuses and cultured in IMDM medium. For the reprogramming protocol, the isolated fibroblasts were seeded 24 h before lentivirus transduction on 0.1% gelatin-coated wells of a 6-well plate at a density of 2 × 10⁴ cells/well. A fresh medium with lentivirus stock was added to the culture medium, incubated overnight, and replaced by a lentivirus-free medium the following day (transduction was considered day 0). On day 5, the cells were harvested with TrypLE and transferred into an MEF feeder, inactivated by mitomycin-C. MEF feeder layers with reprogrammed cells were cultured in iPSC medium, supplemented with 10 ng/ml bFGF [cat. 100-18B, Peprotech] or ESGRO^® Recombinant Mouse LIF Protein [cat. ESG1106—Millipore] associated or not with 2i (1 mM PD0325901 [cat. 444968, Sigma] and 3 mM CHIR99021 [cat. 361571, Sigma]) at 38.5°C, 5% CO₂, and 5% O₂ in a humidified atmosphere. Experimental groups were denominated based on their supplementation, such as bFGF, bFGF2i, LIF, and LIF2i. Additionally, biPSCs were frozen once they reached ∼70% confluency in cryopreservation medium consisting of 90% iPSC medium and 10% (vol/vol) DMSO.

MUSIi013-A cells were transduced with lentiviral particles carrying the anti-CD19 in the presence of 4 μg/mL hexadimethrine bromide (Sigma-Aldrich, St. Louis, MO, USA). Single cells were seeded on irradiated human foreskin fibroblasts in NutriStem medium supplemented with SMC4 small-molecule cocktail inhibitors containing PD0325901 (Sigma Aldrich, St. Louis, MO, USA), CHIR99021, Thiazovivin, and SB431542 (STEMCELL Technologies, Vancouver, BC, Canada) [21 (link)]. Emerging single-cell colonies were manually picked up and cultured on Matrigel-coated plates and underwent full iPSC characterization. Clone 5-19H10 (CAR19-NK/iPSC) and its parental cells (WT-NK/iPSC) were used in this study.

All chemicals were dissolved in E3 medium and administered by immersion from 48 to 74 hpf. Vegf signalling was inhibited using 25 nM tivozanib (AV-951, AVEO Pharmaceuticals); vehicle control groups were exposed to 0.0025% dimethyl sulfoxide (DMSO). mTOR signalling was inhibited using 2-2.5 µM rapamycin (Sigma-Aldrich); vehicle control groups were exposed to 0.2-1% DMSO. MEK signalling was inhibited using 7.5-10 µM PD0325901 (Anastasaki et al., 2012 (link)) (Sigma-Aldrich); vehicle control groups were exposed to 0.75-1% DMSO. p38 MAPK signalling was inhibited by 25 µM SB 203580 (Sigma-Aldrich). Nos inhibition was achieved by incubation with 0.5 mM L-NAME (Sigma-Aldrich) diluted in E3 medium.

Human CRC cell lines used in this study were HCT-116 (CVCL_0291), HT-29 (CVCL_0320) and SW-620 (CVCL_0547). The cell lines were directly obtained from NCI. No mycoplasma testing was done in-house. Cells were routinely cultured in 1X RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich), 2 mM l-Glutamine (Sigma Aldrich) and 100 U/mL Penicillin–Streptomycin (Thermo Fisher Scientific). All cells were maintained at 37 °C with 5% CO₂ and 80% relative humidity and passaged according to in-house protocols (see Supplementary Methods). Cells used in experiments never exceeded passage 21.
Drugs used in screens were olaparib (Selleckchem), oxaliplatin (Selleckchem), palbociclib (Selleckchem), PI-103 (Selleckchem), PD0325901 (Sigma Aldrich), 5-fluorouracil (5-FU, Sigma Aldrich) and 5Z-7-Oxozeaenol (Enzo Life Sciences). Assay reagents used in screens were CellTiter-Glo 2.0 Assay (Promega), CellTiter-Glo 3D Cell Viability Assay (Promega), CellTox Green Cytotoxicity Assay¹⁷ (Promega) and NucView 488 Caspase-3 Substrate¹⁸ (Biotium).

Embryos from CD1 (Charles River laboratories, France) intercrosses were recovered at E2.75 and at E3.25 (expanding blastocyst) by flushing oviducts or uteri in M2 (Millipore), washed twice and cultured inside their intact zona pellucida in 400 µL KSOM (Millipore) in Nunc 4-well plates until the stages of interest at 37 °C, 8% CO₂. For treatment, we used FGF receptor inhibitor PD173074 (100 nM, Sigma Aldrich), MEK inhibitor PD0325901 (1μM, Sigma Aldrich), flavopiridol (1μM, Selleckchem), MG132 (10 μM, Sigma Aldrich) and FGF4 (1 μg/ml, R&D systems) supplemented with heparin (1 μg/ml, Sigma Aldrich). Experiments shown in Figs 2(A–C) and 3 were performed in parallel using same batches of embryos. All experiments were conducted according to the French and European regulations on care and protection of laboratory animals (EC Directive 86/609, French Law 2001–486 issued on June 6, 2001) and were approved by the Institut Pasteur ethics committee (n° 2012–0011).