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. 2020 Nov 11;10:19525. doi: 10.1038/s41598-020-76076-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Vimentin is critical for mESC neuronal differentiation. (a) mESCs differentiated by the addition of retinoic acid (1 µg/ml). Number of cells expressing Oct4 after 4 days of differentiation was observed. Cells were fixed and immuno-stained for Oct4 (green) and Vimentin (red). The white arrows in the image points out that after 4 days WT cells doesn’t have OCT4 expression in the nucleus whereas vim KO cells have. The graph was plotted for percentage of cells expressing Oct4. Error bars represent standard deviation. (b) Whole genome RNA seq data comparison between Wildtype and Vimentin KO using Matlab software. Heat map illustrates that Vimentin Knockout does not affect pluripotent cells (first column on the left) while differentiated (by retinoic acid − 1 µg/ml) cells are affected in their gene expression patterns (right column). (c) Neuronal development genes downregulated in vimentin knockout cells are shown separately using a heat map. (d) EBs differentiated for 4 days were seeded on a different plate for neuronal progenitor cell (NPC) differentiation. Confocal images of the NPCs stained with TUJ1 (green), Vimentin (red) and nucleus (white) in vimentin wildtype cells. Statistics for (1) percentage of cells having a caged vimentin during division and (2) the expression of TUJ1 and Vimentin in the NPCs after division are shown. The error bars represent standard deviation. Statistics were done using two tailed student t-test. (e) Images of the misfolded protein Synphilin-GFP (green) getting asymmetrically partitioned along with Vimentin-RFP (red) in the KO complemented (top panel) and symmetrically partitioned (bottom panel) in the neuronal precursor cells. (f) Confocal images of NPCs stained with neural marker TUJ1 (green), Vimentin (red) and nucleus (white) in Vimentin Wildtype and Vimentin KO cells. (g) statistics for the expression values of vimentin and TUJ1 after division. D1 and D2 are Daughter 1 and Daughter 2 respectively. P < 0.01. Statistics for number of divisions with vimentin in cage/filamentous was also plotted (right panel). (h) Statistics for the percentage of symmetric inheritances of aggregates in Knockout complemented and Vimentin Knockouts. Statistics showing percentage of cells positive for TUJ1 marker that contain vimentin cage (middle panel) and Statistics showing percentage of cells having TUJ1 expression in wildtypes and knockouts (500 cells/replicate) and the error bars represent standard deviation (P < 0.01). (right panel).