Immunoprecipitation of endogenous RNA/protein Selinexor complexes were performed as previously described.8, 10 Total proteins were extracted in radioimmunoprecipitation assay buffer. Cytoplasmic and nuclear lysates were prepared with the subcellular proteome extraction kit (Calbiochem). Immunoblotting analysis was performed with specific antibodies (Supporting Table 2). A detailed immunohistochemistry (IHC) protocol of paraffin-embedded

sections is provided in the Supporting Materials. We found that activated HSCs (α-SMA+ cells) strongly expressed HuR in surgically resected liver samples from patients with alcoholic (Fig. 1A) and HCV cirrhosis (Fig. 1B). Similarly, activated HSCs expressed HuR in the nucleus of liver sections from two animal models of induced fibrosis—BDL mice (Fig. 1C) and rats treated with CCl45 (Fig. 1D)—suggesting that HuR could play a role during HSC activation. To confirm the role of HuR in liver

fibrosis, we silenced HuR in vivo in BDL mice. Thus, mice were injected in the tail vein with an HuR-specific or control shRNA at 0 hours as well as days 3 and 6 after BDL, then sacrificed 9 days after BDL. HuR silencing was confirmed by qPCR and western blotting in whole liver extracts (Supporting Fig. 1A,B) and, specifically, in HSCs by IHC (Supporting Fig. 1C). HuR silencing resulted in reduced histological liver damage, as observed by hematoxylin and eosin (H&E) staining (Fig. 2A) and decreased alanine aminotransferase and bilirubin serum levels (Supporting Fig. 1D,E). Notably, fibrosis development in these mice was significantly attenuated, Tyrosine Kinase Inhibitor Library solubility dmso as shown by reduced collagen deposition (Fig. 2B), α-SMA expression (Fig. 2C), and col1a1, α-SMA, and TGF-β mRNA levels (Fig. 2D). HuR silencing

also led to reduced protein oxidation (Fig. 3A and Supporting Fig. 1F,G), proliferation (Fig. 3B), macrophage infiltration (Fig. 3C), and lower expression of genes involved in inflammation (iNOS [inducible nitric oxide synthase], IL-1α [interleukin-1α], and TNF-α [tumor necrosis factor alpha]) and infiltration (MCP-1 [monocyte chemoattractant protein-1], F4/80, ICAM-1 [intracellular adhesion 上海皓元医药股份有限公司 molecule 1], MMP9 [matrix metalloproteinase 9], and Actin) (Fig. 3D). Altogether, our results suggest that HuR plays a crucial role in the pathogenesis of cholestatic liver injury. The above data suggest that HuR could be regulating HSC activation and fibrosis development, either directly and/or indirectly, by a decrease in liver damage and inflammation. To characterize the effect of HuR in HSC activation only, we examined its expression in primary HSCs isolated from sham and BDL mice 9 days after surgery. HuR mRNA levels increased in HSCs isolated from BDL mice, correlating with HSC activation, as observed by the induction of α-SMA mRNA expression (Fig. 4A). Total, cytoplasmic, and nuclear HuR protein levels were also up-regulated (Fig. 4B).