Nih 3t3 flp in

T-Rex-293 (Invitrogen), IMCD3 Flp-In (IMCD3; American Type Culture Collection; gift of Peter Jackson), Phoenix A (PhA; Indiana University National Gene Vector Biorepository), and 293FT cells were cultured in DMEM high glucose (Sigma-Aldrich; supplemented with 10% cosmic serum, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine). NIH 3T3 Flp-In (Thermo Fisher) cells were cultured in DMEM high glucose media (D5796; Sigma) with 10% Bovine calf serum (BCS; Sigma-Aldrich), 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine. 3T3-L1 cells (gift of Peter Michaely, UTSW) were cultured in DMEM high glucose (Sigma-Aldrich) media with 10% fetal bovine serum, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, 4.5 mM glutamine, and 8 ng/ml biotin. Cell lines were tested negative for Mycoplasma using the Mycoplasma PCR Detection Kit (Genlantis). Transfection of plasmids was done with Polyfect (QIAGEN) or polyethylenimine (PEI) max. Stable cell lines were generated by retroviral infection or transfection. In many cases, stable lines were flow sorted and further selected for GFP. Control and Tulp3 ko MEFs were from embryonic day 13.5 embryos (Legue and Liem, 2019 (link)). Immortalized WT and Arl13b mutant MEFs were gifts from Tamara Caspary (Larkins et al., 2011 (link); Gigante et al., 2020 (link)).

Kif3a^-/-; Kif3b^-/- double knockout 3T3 cells were generated and are described in.¹² (link) They were generated via CRISPR/Cas9-mediated knockout from the parental cell line NIH 3T3 Flp-In (Mus musculus embryonic fibroblast; RRID: CVCL_U422) purchased from Thermo Fisher Scientific. The Kif3a^-/-; Kif3b^-/- 3T3 cells were cultured in DMEM (Corning) supplemented with 10% Fetal Clone III (Cytiva Hyclone) and 4 mM L-glutamine (Alfa Aesar) at 37°C and 5% CO₂. Subculturing was performed to keep cells between 4 to 80% confluent. The cells have a male karyotype, but since we are studying fundamental processes in motor function and ciliogenesis and these mechanisms are found in all cells, we have no reason to expect differences based on sex. The presence of the double knockout is regularly re-authenticated via the rescue of ciliogenesis by co-transfection with plasmids driving the expression of KIF3A and KIF3B. All cells in the lab are periodically tested for mycoplasma contamination via a commercially available PCR test (ATCC).