Feline Lindhout

3 Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons 93 Electrophysiology Before the start of each experiment, a 12 mm coverslip with Centrione-B treated or control human iPSC-derived neurons (paired cultures of 7–14 days after plating) was placed under the microscope. In the recording chamber, carbogenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF, in mM: 126 NaCl, 3 KCl, 2.5 CaCl2, 1.3 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 20 glucose; with an osmolarity of ~310 mOsm/L) was continuously perfused at a rate of approximately 1 ml/min. An extra medium refreshment was conducted one day before recording, to prevent changes in excitability due to acute differences in extracellular osmolarity (Pasantes-Morales 1996). ACSF was warmed before entering the bath and the temperature was maintained at 30-32 °C. Recording pipettes pulled from thick-walled borosilicate glass capillaries (World Precision Instruments) had a resistance of 4-7 MΩ and were filled with internal solution (in mM: 140 K-gluconate, 4 KCl, 0.5 EGTA, 10 HEPES, 4 MgATP, 0.4 NaGTP, 4 Na2-Phosphocreatine; with pH 7.3 and osmolarity 295 mOsm/L). To facilitate visualization of cells during the experiment and post hoc, the internal solution was supplemented with 30 µM Alexa 568 (Thermo Fisher Scientific) and biocytin, respectively. We selected individual neurons with a 60x water immersion objective (Nikon NIRApochromat; NA1.0) and performed whole-cell somatic patch clamp recordings. During the recordings in both voltage and current clamp, cells were kept at a holding potential of -60 mV. Recordings were acquired with an Axopatch 200B amplifier (Molecular Devices) using pClamp 10 software. Data was analyzed with Clampfit 10.7 software and custom- written MATLAB scripts. Mass spectrometry To obtain sample preparations for mass spectrometry (TMT labeling), biological replicates of control and Centrinone-B treated human iPSC-derived neurons were harvested at three different time points (days 1, 3, and 7) of differentiation with lysis buffer (8 M urea, 50 mM ammonium bicarbonate (Sigma), EDTA-free protease inhibitor cocktail (Roche)). Lysates were sonicated on ice using Bioruptor (Diagenode) and cleared by centrifugation at 2500g for 10 minutes. Protein concentration was determined using Bradford assay. From each condition 100 µg of proteins were reduced (5 mM DTT, 55˚C, 1 hour), alkylated (10 mM Iodoacetamide, 30 minutes in the dark) and sequentially digested by LysC (Protein- enzyme ratio 1:50, 37˚C, 4 h) and trypsin (Protein-enzyme ratio 1:50, 37˚C, overnight). After overnight digestion, samples were acidified with formic acid (final concentration 3%) and resulting peptides were then desalted using Sep-Pak C18 columns (Waters). Samples were subjected to sTable isotope TMT-6plex labeling according to manufacturer’s instruction (Thermo Fisher Scientific). In brief, peptides were resuspended in 80 µl of 50 mM HEPES buffer, 12.5% ACN with pH 8.5 while TMT reagents were dissolved in 50 µl anhydrous ACN. For labeling, 25 µl of each dissolved TMT reagent were added to the correspondent sample according to the following scheme: Control human iPSC sample, day 1 = TMT-126 / Centrinone-B human iPSC sample, day 1 = TMT-129 Control human iPSC sample, day 3 = TMT-127 / Centrinone-B human iPSC sample, day 3 = TMT-130