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. 2023 Feb 15;37(4):864–876. doi: 10.1038/s41375-023-01838-8

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© The Author(s) 2023

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — A Experimental overview created with BioRender.com for the SUMO2 target identification upon 4 or 20 h of 5-Aza-2’ treatment in U2OS and Namalwa cells. U2OS and Namalwa cells stably expressed His10-SUMO2. His10-SUMO2 targets were enriched via Ni-NTA pulldown. Proteins were trypsin digested and prepared for LFQ mass spectrometry. Four replicates were prepared per condition and analyzed by nano flow LC-MS/MS. B, C respectively show volcano plots visualizing all identified SUMOylated proteins in U2OS His10-SUMO2 upon 4 or 20 h of 5-Aza-2’ treatment (1 µM) compared to control. His-SUMO2 target proteins were enriched via Ni-NTA pulldown, followed by trypsin digestion and LFQ mass spectrometry, peptides were identified by LC-MS/MS. Dashed lines represent cut off at a foldchange of two (log2 = 1) and p-value of 0.05 (-log10 = 1.3) (n = 4). D STRING network analysis of enriched SUMOylated proteins upon 4 h of 5-Aza-2’ treatment with Cytoscape Software at a confidence score of 0.4. The interaction network visualizes fold change via node color as indicated in the scale bar (fold change of: 0.59–12.17) and significance indicated with node size. E MCODE was used to extract the most interconnected clusters from the STRING network analysis in D. Cluster 1 represents proteins involved in chromosome organization and negative regulation of transcription. F Cluster 2 represents DNA damage response proteins. G Cluster 3 represents proteins involved in SUMOylation. H Bar-graph visualizes Gene Ontology enrichment analysis of proteins SUMOylated upon 4 h of 5-Aza-2’ treatment, for GO molecular functions, GO Cellular components and GO Biological processes compared against the reference humane proteome. Only pathways significantly enriched and with a fold enrichment of more than 20 are shown. I STRING network analysis of enriched SUMOylated proteins upon 20 h of 5-Aza-2’ treatment with Cytoscape software at a confidence score of 0.4. The interaction network visualizes fold change via node color as indicated in the scale bar (fold change of: 0.63–8.2) and significance indicated with node size. J MCODE was used to extract the most interconnected clusters from the STRING network analysis in I. Cluster 1 represents proteins involved in methylation. K Bar-graph visualizes Gene Ontology enrichment analysis of proteins SUMOylated upon 20 h of 5-Aza-2’ treatment, for GO molecular functions. Only pathways significantly enriched and with a fold change of more than 20 are shown. Source data are provided as Source data file_MS or _GeneOntology.