RNA-seq analysis of gene expression profile of wildtype and Oxsr1 knockout HEK293T cells

Our study aims to identify the potential signaling pathways regulated by osmosensor OXSR1. Animals activate regenerative processes to repair injuries and restore homeostasis following tissue damage. A central question in regeneration is how damage signals are sensed and translated into regenerative growth. Tissue injuries lead to the release of intracellular contents and bodily fluids and disturb osmotic balance. However, the role of osmolarity in regeneration remains largely unexplored. Using Drosophila and mouse intestine, and samples from inflammatory bowel disease (IBD) patients, we identify a key role for the osmolarity-sensing WNK-OXSR1 signaling pathway in intestinal regeneration. Mechanistically, upon intestinal injury, OXSR1 phosphorylates the RhoB GTPase at threonine 37, disrupting its interaction with ARHGAP17, increasing GTP-bound RhoB, enhancing F-actin polymerization, and activating YAP to drive regeneration. We further show that pharmacologically inhibiting WNK or OXSR1 reduces the oncogenic potential of intestinal regeneration. These findings reveal osmolarity as a critical damage signal in regeneration and position WNK-OXSR1 as a potential therapeutic target to promote intestinal repair. Overall design: RNA was extracted using Trizol Reagent (Invitrogen) and purified using RNeasy Mini Kit (Qiagen) following the kit manual. Library construction, sequencing and bioinformatics analysis were performed by MegaGenomics Inc. (Beijing, China). The P value was calculated by Deseq2 (v1.26.0). The differentially expressed genes (DEGs) were defined as adjusted p-value < 0.05 and |log2(FoldChange)|>0.5 Three biological replicates were performed for WT and Oxsr1 KO HEK293T cells.