Journal: bioRxiv
Article Title: A chemical inducer of ribophagy limits the toxicity of ALS-related arginine-rich peptides
doi: 10.64898/2026.03.15.711847
Figure Lengend Snippet: A. Structure of SALSa functionalised with an alkyne group (SALSa alk ). B. Activity of SALSa alk in rescuing toxicity in U2OS PR97 cells. Plotted is the viability normalised against dox-treated cells (N = 3). C. Microscopy images of U2OS cells treated with 10 µ M SALSa alk for 2 h. Cells were stained with Hoechst 3342 (blue) to visualise nuclei, SALSa alk was visualised by Click chemistry coupling of Alexa 6 fluorophore (red). Counter-staining with antibodies (green) was done to visualise lysosomes (LAMP1) or nucleoli (NPM). Scale bar as indicated. D. Volcano plot of the proteomic analysis of SALSa alk interacting proteins. Horizontal red line indicates the p-value threshold (<0.05), while the vertical red line marks a 2x fold-change. Yellow dots represent proteins in the data set belonging to the ‘rRNA processing’ Gene Ontology Biological Process term, the most significantly enriched in the data set. E. Enrichment analysis of Gene Ontology Biological Processes among the proteins enriched at least 2-fold in the SALSa alk pull-down sample compared to the mock pull-down. The analysis was done using STRING. F. Northern blotting of rRNA intermediates in U2OS cells treated for 24 h with 10 µ M SALSa. Total cellular RNA was resolved using agarose gel electrophoresis, blotted onto nitrocellulose membrane, and biotinylated probes directed to Internal Transcribed Spacers (ITS1 and ITS2) in rRNA primary transcript (see schematic) were hybridized. The image of GelRed-stained 28S mature rRNA is provided as a loading control. G. As in F, but total cellular RNA was resolved on a polyacrylamide gel to enable the analysis of 5.8S rRNA maturation intermediates. H. Analysis of 5.8S rRNA maturation in U2OS cells treated for 24 h with 10 µ M SALSa using a circularised RNA RT-PCR assay. An scheme illustrating the assay is provided to the left of the data. Total RNA was circularised with T4 RNA ligase, followed by reverse transcription with an internal 5.8S primer and end-point PCR on the resulting cDNA. Reactions were resolved by agarose gel electrophoresis.
Article Snippet: Samples were electrophoresed on a Bis-Tris gel and electroblotted onto a nitrocellulose membrane (Bio-Rad, 1704270).
Techniques: Activity Assay, Microscopy, Staining, Northern Blot, Agarose Gel Electrophoresis, Membrane, Control, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription
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