Feline Lindhout

3 Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons 91 Technologies), anti-mouse Alexa 488 (A11029, Life Technologies), anti-mouse-IgG1 Alexa 488 (A21121, Life Technologies), anti-rabbit Alexa 568 (A11036, Life Technologies), anti- mouse Alexa 568 (A11031, Life Technologies), anti-mouse-IgG1 Alexa 594 (A21125, Life Technologies), anti-mouse-IgG2a Alexa 594 (A21135, Life Technologies), anti-chicken Alexa 647 (A21449, Life Technologies), anti-rabbit Alexa 647 (A21245, Life Technologies) anti-mouse Alexa 647 (A21236, Life Technologies), anti-mouse-IgG2a Alexa 647 (A21241, Life Technologies), phalloidin Alexa 647 (A22287, Life Technologies). Immunofluorescence For centrosomal stainings, cells were fixed for 10 min in methanol at -20°C. For all other experiments, cells were fixed for 10 min in PBS with 4% formaldehyde/4% sucrose (for neurons) or 10 min in PBS with 4% formaldehyde (for non-neuronal cells) at room temperature. After fixation, cells were washed three times with PBS. Next, fixed cells were sequentially incubated with primary and secondary antibodies diluted in gelate dilution buffer (GDB; 0.2% BSA, 0.8 M NaCl, 0.5% Triton X-100, 30 mM phosphate buffer, pH 7.4). Cells were washed three times with PBS after every antibody incubation, and coverslips were mounted using Vectashield mounting medium (Vector laboratories) with or without DAPI. Microscopy Confocal microscopy of fixed cells was performed on a LSM700 confocal laser-scanning microscope (Zeiss) equipped with the following objectives: 1) a Plan-Apochromat 63x NA 1.4 oil DIC; 2) a EC Plan-Neofluar 40x NA 1.3 Oil DIC; and 3) a Plan-Apochromat 20x NA 0.8 objective. Dual-color gated STED (gSTED) imaging was performed on a Leica TCS SP8 STED 3X microscope using a HC PL APO 100x/ N.A. 1.4 oil immersion STED WHITE objective. The 488, 590 and 647 nm wavelengths of pulsed white laser (80MHz) were used to excite Alexa 488-labeled γ-Tubulin, Alexa 594-labeled Centrin and Alexa647- labeled Trim46, respectively. Alexa 594 and Alexa 647 were depleted with the 775 nm pulsed depletion laser and an internal Leica HyD hybrid detector (100% gain) with a time gate of 0.3 ≤ tg ≤ 6ns was used. Spinning-disk confocal microscopy for live-cell imaging was performed on an inverted microscope Nikon Eclipse Ti-E, which was equipped with a spinning disk-based confocal scanner unit (CSU-X1-A1, Yokogawa), an ASI motorized stage with the piezo plate MS-2000-XYZ (ASI), and the perfect focus system (Nikon). We used the following cameras: 1) a Photometric Evolve Delta 512 EMCCD camera controlled by the MetaMorph 7.8 software (Molecular Devices), or 2) a Photometric PRIME BSI sCMOS camera (version USB 3), controlled by the MetaMorph 7.10 software (Molecular Devices). We used the 491 nm 100mW Calypso (Cobolt) and 561 nm 100mW Jive (Cobolt) lasers as light sources. For imaging of GFP-tagged proteins we used an ET-GFP filter set (49002, Chroma), for imaging of proteins with tag-RFP we used an ET-mCherry filter set (49008, Chroma). For live-cell imaging with photoablation, we used an ILas system (Roper Scientific France/PICT-IBiSA, Institut Curie, currently Gataca Systems) mounted on the