Feline Lindhout

2 38 by unsupervised clustering, and identified six clusters with distinct expression profiles (Fig 2D,E; Table 5). Proteins in clusters 1, 2, and 3 were upregulated, and proteins present in cluster 4, 5, and 6 were downregulated during early neuronal development. Moreover, many proteins within specific clusters showed overlap in functions (Fig 2D,E; Table 5). Cluster 1 contains proteins that show a slight increase from day 1 to day 7. GO enrichment analysis revealed enrichment of several terms related to neuronal differentiation and intracellular transport mechanisms, which reflects cell-autonomous remodeling of molecular processes (Fig 2D, Table5). One of the upregulated proteins in cluster 1 is KLC1, a subunit of the microtubule motor protein, which was found to be required for neuronal differentiation from human embryonic stem cells (Killian et al. 2012). The AIS protein Trim46, which is known to regulate neuron polarity and axon specification by controlling microtubule organization during development, was also found in this cluster (van Beuningen et al. 2015). Furthermore, this cluster contains Camsap1, Camsap2 and Camsap3, proteins which localize to the minus ends of microtubules to stabilize them, thereby regulating neuronal polarity (Jiang et al. 2014). Proteins in cluster 2 present a considerable increase in relative expression from day 3 to day 7, which coincides with the onset of axon formation and development (stage 3). Accordingly, enriched GO terms include proteins associated with neuronal development, axonogenesis, and other axon-related mechanisms (Fig 2D). Similarly, the GO terms neuronal development, axon, and synapse were also upregulated during differentiation of immortalized human neural progenitor cells (Song et al. 2019). Among the highly upregulated proteins in this cluster are several members of the Septin family: neuronal- specific Sept3, Sept5, and Sept6 (Table 5). Although mechanistic insights remain unclear, emerging evidence implicates Septins as potential factors for establishing neuronal polarity (Falk, Boubakar, and Castellani 2019). Septins interact with actin and microtubule networks and could affect neuronal polarity by regulating cytoskeleton dynamics (Spiliotis 2018; Falk, Boubakar, and Castellani 2019). Sept6 specifically is suggested to play a role in axonal filopodia formation as well as in dendritic branching, and its increased expression coincides with axonal outgrowth (Cho et al. 2011; Hu et al. 2012). Moreover, examples of proteins with the highest relative expression in this cluster are DCX, Tau, Ncam1, Basp1, Snap91, and Syt1, which are generally considered to be neuronal differentiation and polarization markers (Table 5). These data confirm the neuronal identity of the human iPSC-derived cells, and the presence of cellular machinery involved in axon development. Cluster 3 represents proteins with increased expression from day 1 to day 3, and minimal changes in expression from day 3 to day 7. This cluster comprises proteins enriched in GO terms that are associated with cell metabolism and (re)localization of intracellular and extracellular components, which correspond to substantial changes in the cellular proteome (Fig 2D). Proteins in this cluster that show differential expression from day 1 to day 3 include Sox4 and Sox11, both members of the SoxC transcription factor family (Table 5). These factors are involved in neurogenesis and their expression induces subsequent expression of neuron-specific genes (Kavyanifar, Turan, and Lie 2018). Also represented in this cluster are Arpc2 and Arpc4, subunits of the Arp2/3 complex. The Arp2/3 complex mediates actin polymerization and is required for