Fig. 4

Identification of CLUH proximal proteins using BioID in HCT116 cells. A Schematic representation of the BioID experimental design using HCT116 cells stably expressing the BioID2 protein fused to the mouse CLUH (mCLUH) or GFP proteins. The proximity labeling is performed for 24 h in the presence of 50 μM biotin in the culture medium. Biotinylated proteins, from both the specific (BioID2-CLUH) and control (GFP-BioID2) samples, are isolated using streptavidin-coupled magnetic beads and identified by LC MS/MS. B Table summarizing the MS protein identification from the BioID experiment in HCT116 cells. Total number of proteins identified by Mascot software with a FDR below 1%. The five proteins with the highest specific spectral counts in the BioID2-mCLUH sample are shown. Biological replicate samples are numbered from #1 to #3. The full datasets and analysis are available in Table S3. C Volcano plot showing the global enrichment of proteins in BioID2-CLUH versus the GFP-BioID2 control. The x-axis shows the log2 fold change (FC), and the y-axis shows the −log10 of the false discovery rate (n=3), obtained using SAINTexpress software [26]. Significantly enriched proteins are shown in red and are defined by a fold change greater than two and a FDR < 0.1 (shown as dashed red lines). The five proteins with the highest spectral count (shown in B) are labeled and identified with a green circle. D Manhattan plot illustrating the gene ontology and pathway enrichment analysis of proteins identified in BioID experiment, generated using G:profiler tool [32]. The functional terms, associated with the protein list, are grouped in four categories: GO: MF (Molecular Function), GO: CC (Cellular Component), GO: BP (Biological Process), and KEGG pathways. The y-axis shows the adjusted enrichment p values in the negative log10 scale. The circle sizes are in accordance with the corresponding term size (i.e. larger terms have larger circles) and terms from the same GO subtree are located close to each other on the x-axis. The most significantly enriched terms are labeled. E Visualization of the functional interaction network of CLUH proximal proteins identified by BioID, generated with the Cytoscape StringApp [33]. The proteins have been grouped according to three most represented functional categories: “Cytoskeleton related”, “Translation” and “Mitochondrial proteins”. The confidence score of each interaction is mapped to the edge thickness and opacity. The size of the node relates to the enrichment in log2 fold change (log2FC) over the BioID-GFP background control. The protein abundance in the BioID2-CLUH sample is illustrated by a color scale and corresponds to the specific spectral count normalized to the protein size. Proteins with mitochondrial targeting sequences (MTS) according to Uniprot database are highlighted in red