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. 2021 Jan 28;14:612560. doi: 10.3389/fncel.2020.612560

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Copyright © 2021 Červenka, Tylečková, Kupcová Skalníková, Vodičková Kepková, Poliakh, Valeková, Pfeiferová, Kolář, Vaškovičová, Pánková and Vodička.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Influence of the VEGF pathway on growth and survival of NSCs in culture. (A) SWATH-MS results show an increase of protein abundances of neuropilin-1 and catenin β-1 during S and BG differentiation. Symbols denote significant difference (p < 0.05, moderated t-statistic from limma) of a given group vs. day 0 (#), or day 7 (†) of the same differentiation protocol. (B) Expression of catenin β-1, HIF1-α, neuropilin-1, and VEGF121 during both differentiations detected by immunoblotting. rVEGF121 and rVEGF165—recombinant proteins. Symbol + denotes mix of proliferating and differentiated NSCs used as a positive control. (C) Cell growth is depicted as a change in confluence (acquired and analyzed on an IncuCyte incubator microscope) in time. Data from 3 independent experiments with 6 wells in each experimental condition are shown. Full lines indicate a smooth average over 6 wells for a given condition, dotted lines data from individual wells. (D) The area under the growth curve was calculated independently for each well and is displayed as mean (dot) and confidence interval (line) for each of the three experiments. Statistical significance was calculated by fitting a linear mixed model of VEGF and EGF/FGF2 supplementation within each experiment and is denoted with ^*** (Tukey adjusted p < 0.001) in the given pairwise comparison. (E) Cell damage, as indicated by SYTOX Green positivity. After 8 days of cultivation, cells were treated with vital and dead cell staining and captured images were analyzed in Fiji. Data from two independent experiments with 3 wells per condition and 3 images per well are shown. Statistical significance was calculated from a linear mixed model of VEGF and EGF/FGF2 supplementation within each experiment, with Tukey-adjusted p-values for pairwise comparison and denoted with ^*** (p < 0.001). (F) SWATH-MS results show an increase of protein abundance of caspase-3 at day 7 in both differentiation protocols. Symbols denote significant difference (p < 0.05, moderated t-statistic from limma) of a given group vs. day 0 (#). (G) Detection of activated caspase-3 during S and BG differentiation confirmed its increase at days 7 and 14 in comparison to day 0. Immunoblots shown in (B,G) are representative images from two biological replicates. Corresponding loading controls (GAPDH and ATPα) are shown in Figure 7B.