Generation and Characterization of Mutant MEFs

Generation of wild-type, Fuzzy^−/− and ArhGap35^D34/D34 MEFs was previously described (Seo et al., 2011 (link); Stewart et al., 2016 (link)). MEFs were plated on 15 mm glass coverslips (Thermo Fisher Scientific) coated with rat Collagen I (Life Technologies) at 10⁵ cells per well in 12-well plates (Sarstedt). DMEM/F12 growth medium was supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA) and 1% penicillin/streptomycin (all from Wisent). After 24 h, cells were starved in serum-free medium for another 24 h to induce ciliogenesis and then treated with actin polymerization inhibitors for 8 h. Fasudil was diluted to 50 µM in PBS, Y27632 diluted to 1 µM, and cytochalasin D to 0.5 µM in DMSO (all inhibitors from Sigma-Aldrich). Inhibitor concentrations were optimized to ensure high cell viability using Cell Counting Kit - 8 reagent (Sigma-Aldrich) according to manufacturer recommendations. At the end of the incubation period, cells were washed with DMEM and fixed with 4% paraformaldehyde in PBS (PFA/PBS) for 15 min after four 3-min washes with PBS. For colocalization experiments, both Fuzzy^−/− and ArhGap35^D34/D34 MEFs were grown under similar conditions. Human embryonic kidney HEK293T cells were grown in 100 mm Petri dishes in DMEM supplemented with 10% FBS and 1% streptomycin/penicillin until ready for transfection.