Feline Lindhout

3 90 to Neuron Differentiation-XF Medium (ax0032, Axol Bioscience), and differentiated neurons were next maintained in Neuronal Maintenance-XF Medium (ax0034, Axol Bioscience). Primary rat hippocampal neuron, HeLa and IMCD3 cell culture Dissociated rat hippocampal neuron cultures were prepared as previously described (Lindhout et al. 2019). In brief, neurons were obtained from embryonic day 18 rat pups, plated on poly-l-lysine/laminin-coated coverslips (100k/well), and kept in Neurobasal medium (NB) supplemented with 2% B27, 0.5 mM glutamine, 16.6 μM glutamate, and 1% penicillin/streptomycin at 37°C with5% CO2. HeLa or IMCD3 cells were plated on plastic for expansion and 18 mm coverslips for experiments, and kept in DMEM/Ham’s F10 (50%/50%) medium supplemented with 10% FCS and 1% penicillin/streptomycin at 37°C with 5% CO2. Pharmacological treatments For centriole loss experiments, cells were treated with Centrinone-B (500 nM; HY-18683, MedChemExpress) 1 hour after plating and treatment was continued with every following medium change to prevent unintentional wash-out. For growth cone experiments, if indicated, cells were treated for 24h with 20 nM Nocodazole (Sigma-Aldrich, M1404) prior to fixation. Lentiviral infection Constructs expressed by lentiviral infections in this study are FUGW-GFP (Addgene #14883, (Lois et al. 2002)) and Marcks-tagRFP-T-pIres-GCN4-MacF18. Cloning of Marcks- tagRFP-T-pIRES-GCN4-MacF18 is described previously (Yau et al. 2014). In short, the construct was subcloned into the lentiviral vector pSIN-TRE-mSEAP-hPGK-rtTA2sM2 (kind gift from Dr. Didier Trono, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland) in which the PGK promotor was substituted by the neuron-specific synapsin promotor. Lentiviral particles were generated as described previously (Yau et al. 2014). Cells were transduced with lentivirus 2 hours after plating. The tetracycline-dependent expression was induced two days before imaging by supplementing the medium with 500 ng/ml doxycycline. Antibodies The following primary antibodies were used in this study: mouse-IgG1 anti-γ-Tubulin (1:500; T6557, Life Technologies), mouse-IgG2a anti-Centrin (1:300; 20H5, Millipore), rabbit anti-Ki67 (1:500; ab92742, Abcam), chicken anti-MAP2 (1:2000; ab5392, Abcam), rabbit anti-Trim46 (1:500; homemade, (van Beuningen et al. 2015)), mouse-IgG1 anti-Nestin (1:200; MAB5326, Millipore), mouse-IgG1 anti-α-Tubulin (1:1000; T-5168, Sigma), rabbit anti-pericentrin (1:500; 923701, BioLegend), mouse-IgG1 anti-AnkG (1:200; 33-8800, Life Technologies), mouse-IgG2b anti-β3-Tubulin (1:400; T8660, Sigma). The following secondary antibodies were used in this study: anti-chicken Alexa 405 (ab175675, Abcam), anti-rabbit Alexa 488 (A11034, Life Technologies), anti-chicken Alexa 488 (A11039, Life