23-02492-RNAseq-NSA 1.25 uM
Description
Centriolar satellites are high-order assemblies, scaffolded by the protein PCM1, that gravitate as particles around the centrosome and play pivotal roles in fundamental cellular processes notably ciliogenesis and autophagy. Despite stringent control mechanisms involving phosphorylation and ubiquitination, the landscape of post-translational modifications shaping these structures remains elusive. Here, we report that necrosulfonamide (NSA), a small molecule known for binding and inactivating the pivotal effector of cell death by necroptosis MLKL, intersects with centriolar satellites, ciliogenesis, and autophagy independently of MLKL. NSA functions as a potent redox cycler and triggers the oxidation and aggregation of PCM1 alongside select partners, while minimally impacting the overall distribution of centriolar satellites. Additionally, NSA-mediated ROS production disrupts ciliogenesis and leads to the accumulation of autophagy markers, partially alleviated by PCM1 deletion. Together, these results identify PCM1 as a redox sensor protein and provide new insights into the interplay between centriolar satellites and autophagy.
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Steps to reproduce
Jurkat cells were treated with vehicle (DMSO) or NSA (2.5 μM) overnight, in three independent experiments, washed with PBS, and snap-frozen on dry ice. RNA extraction (all RNA integrity number > 9.0), library preparation, RNAseq, and bioinformatics analysis were performed at Active Motif (Carlsbad, California, USA). Briefly, 2 µg of total RNA isolated using the NucleoSpin RNA plus Mini Kit for RNA Purification were further sequenced in Illumina sequencing (using NextSeq 500). The paired-end 42 bp sequencing reads (PE42) generated by Illumina were mapped to the genome using the STAR algorithm - DESeq2 software pipeline described in the Data Explanation document. Genes considered differently expressed had an adjusted p-value of less than 0.1. Enrichment analysis of the RNAseq experiment were performed using g:Profiler (version e107_eg54_p17_bf42210) applying a significance threshold of 0.01 and a |log2(fold change)|>1.2.