MEF generation

MEFs were generated from E13.5 embryos resulting from homozygous ROCK1wt and ROCK1nc breeding pairs. The appearance of the mating plug indicated embryonic day 0.5 (E0.5), and 13 days later pregnant females were euthanized by cervical dislocation and uterine horns containing embryos extracted. Briefly, the embryos were dissected from the uterus and washed in PBS. Embryonic heads and livers were removed and used for genotyping. The remaining tissue from all embryos was pooled and roughly chopped before incubation in 0.05% trypsin (1 mL trypsin/embryo) for 10 min at 37°C. Embryos were then further dissociated by trituration in serological pipettes of decreasing size, then counted and plated at 5 × 10⁶ cells in 180 cm flasks. Cells were cultured in DMEM supplemented with 10% FBS, penicillin/streptomycin (100 units/mL, 100 µg/mL, respectively) and maintained at 37°C in 5% CO₂ atmosphere. Cells were split twice before cryopreservation. Typically, ROCK1wt and ROCK1nc MEF cultures proliferated to passage 8–10 before senescing. No differences in proliferation rate or senescence were observed between ROCK1 genotypes.