The RNA-binding protein HuR promotes nonalcoholic steatohepatitis (NASH) progression by enhancing death signaling pathway

Hepatocyte death triggers liver inflammation, liver injury, and fibrosis, which contributes to non-alcoholic steatohepatitis (NASH) pathogenesis. However, whether RNA processing regulates death signaling pathway during NASH progression is not investigated. In this study, we show that HuR, a widely expressed RNA-binding protein, promotes NASH progression by increasing DR5/caspase8/caspase3-mediated hepatocyte death. Cytosolic HuR levels are abnormally elevated in human patients with NASH. Hepatocyte-specific deletion of HuR protects against MCD-induced NASH by decreasing liver steatosis, inflammation and cell death, whereas hepatic overexpression of HuR induces liver injury by increasing DR5-induced hepatocyte death. Furthermore, in primary hepatocytes, HuR deficiency ameliorates PA&TNFα-induced hepatocyte death due to decreased DR5/caspase8/caspase 3 signaling pathway while overexpression of HuR induces hepatocyte death by increasing DR5/caspase8/caspase 3 signaling pathway. Mechanistically, HuR directly binds to 3′-UTR of DR5 transcript and promotes its mRNA stability, contributing to the hepatocyte death during NASH progression. Our data reveal a novel mechanism by which HuR promotes mRNA stability of DR5, which contributes to NASH progression.


Introduction
Non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, liver injury, chronic inflammation and liver fibrosis, which has been recognized as a key step for the development of cirrhosis and hepatocellular carcinoma (HCC) 1,2 . A 'two hit' theory has been proposed to explain NASH pathogenesis 3 . The first hit is hepatic steatosis due to abnormal lipid accumulation in the liver 4,5 . The second hits include oxidative stress, inflammation, cell death and liver injury 3,6,7 . Hepatocyte death is at the center of the second hits because it triggers liver inflammation, liver injury, and fibrosis. Clinical and genetic mouse studies demonstrate that hepatocyte apoptosis plays a key role in NASH progression 8,9,10,11,12 . It has been shown that the ligands for death receptors (e.g., TNFα and TRAIL), death receptors (e.g., FAS and DR5), and the activation of caspases 2, 3, and 8 are abnormally upregulated in the liver of human patients with NASH 11,12,13,14,15 . TRAIL knockout mice are protected from diet-induced NASH 16 . The free fatty acid such as palmitate can induce aggregation of DR5 on the cell membrane and then activate caspase 8/caspase 3 signaling pathway, leading to hepatocyte death 9 . Knockout of caspase 2 or hepatic deletion of caspase 8 attenuates methionine/choline deficient diet (MCD)-induced NASH 12,15 . The treatment of caspase inhibitors have been shown to protect against NASH pathogenesis 17,18 . However, whether RNA processing regulates death signaling pathway during NASH progression is not investigated. ELAV (embryonic lethal/abnormal visual system)/Hu family RNA-binding proteins, such as ELAVL1/HuR/HuA, ELAVL2/HuB, ELAVL3/HuC and ELAVL4/HuD, contain three conserved RNA recognition motifs and specifically bind adenylate-uridylate-rich element (AREs) in the 3′-untranslated region (UTR) of targeted mRNAs, regulating RNA stability and translational efficiency 19,20,21,22 . HuB, HuC and HuD are exclusively expressed in neurons, whereas HuR is ubiquitously expressed, including liver 21 . Several studies have shown that HuR play important roles in the liver de-differentiation, liver development, human hepatocellular carcinoma (HCC) progression, and liver fibrosis 23,24,25 . However, whether HuR regulates NASH progression by modulating RNA processing of death signaling molecules is still largely unknown. 4 In this study, we show that abnormally elevated expression of HuR promotes the pathogenesis of NASH.
Hepatocyte-specific deletion of HuR protects against NASH progression by decreasing liver steatosis, inflammation and cell death, whereas hepatic overexpression of HuR induces liver injury by increasing DR5/caspase8/caspase3-induced cell apoptosis. Furthermore, HuR directly binds to 3′-UTR of DR5 transcript and promotes its mRNA stability, contributing to the hepatocyte death during NASH progression. This study also suggests that HuR might be a novel drug target for the treatment of NASH.

Animal Experiments
Animal experiments were carried out in strict accordance with the Guide for the Care and Use of Laboratory MCD-induced NASH, mice were fed with an MCD (Medicience) for three weeks. Hepatocyte-specific HuR knockout mice were generated by crossing HuR flox/flox mice 26,27 with Alb-Cre mice. Blood samples were collected from orbital sinus. The serum alanine aminotransferase (ALT) activity and TAG levels was measured with an ALT or TAG reagent set, respectively 28 .

Human samples
Human liver samples were collected from the Third Affiliated Hospital of Sun Yat-sen University. The present study was approved by the Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University, and individual permission was obtained using standard informed consent procedures. The investigation conforms to the principles that are outlined in the Declaration of Helsinki regarding the use of human tissues. Samples with a NASH activity score (NAS) ≥ 5 or a NAS of 3-4 but with fibrosis were included in the NASH group and samples with a NAS of 0 were classified as non-steatotic control.

Primary hepatocyte culture and Aadenoviral infection
Primary hepatocytes were isolated from C57BL/6 WT, HuR flox/flox and HuR-HKO mice by liver perfusion with type II collagenase (Worthington Biochem, Lakewood, NJ) and cultured at 37°C and 5% CO2 in DMEM medium supplemented with 5% FBS. Primary hepatocytes from from C57BL/6 WT mice were infected with βGal or Flag-HuR adenoviruses overnight.
Nuclear protein was extracted from the pellets using a high-salt solution (20 mM HEPES, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF and 1 mM Na 3 VO 3 , pH 7.9). The preparation of nuclear and cytosolic protein from primary hepatocytes was using a commercial Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime).

Immunoblotting and RNA-immunoprecipitation
For immunoblotting, total cell lysates, cytosolic and nuclear lysates were immublotted with the indicated antibodies, and visualized using the ECL as shown previously 29 .
Primary hepatocytes were used for RNA-immunoprecipitation. Primary hepatocytes from C57BL/6 WT mice were infected with βGal or Flag-HuR adenoviruses overnight. Primary hepatocytes were also isolated from HuR flox/flox and HuR-HKO mice and cultured overnight. Cell lysates from the above hepatocytes were immunoprecipitated with Flag beads and HuR antibody at 4 ℃ for 2 hours. The subsequential RNA immunoprecipitation (RIP) was performed using the Magna RIP kit (Merck Millipore) according to the manufacturer's instruction. RNA samples retrieved from anti-HuR or Flag-beads were used for RT-qPCR.

Real Time Quantitative PCR (RT-qPCR)
RT-qPCR was performed as shown previously 29,30 . Briefly, total RNAs were extracted using TriPure Isolation Reagent (Roche, Mannheim, Germany), and the first-strand cDNAs were synthesized using random primers and M-MLV reverse transcriptase (Promega, Madison, WI). qPCR was done by using Roche LightCycler 480 real-time PCR system (Roche, Mannheim, Germany). The expression of individual genes was normalized to the expression of 36B4 (a house-keeping gene). Primers for real time qRT-PCR were listed in Supplementary Table 2.

RNA sequencing
RNA-seq were performed as described previously 29,31 . The hepatic mRNA profiles of HuR flox/flox and hepatocyte-specific HuR knockout (HKO) mice (n=3 for each group) fed with an MCD for 3 weeks were generated by deep sequencing using an Illumina Novaseq platform. The mRNA profiles of β-Gal-overexpressing, and HuR-overexpressing primary hepatocytes were also generated by deep sequencing using an Illumina Novaseq 6000 platform. Paired-end clean reads were aligned to the mouse reference genome(GRCm38.p6) with Hisat2 v2.0.5, and the aligned reads were used to quantify mRNA expression by using featureCounts v1.5.0-p3. RNA-seq data that support the findings of this study have been deposited in GEO under accession code GSE152670 and GSE154015.

RNA decay assay
Primary hepatocytes from C57BL/6 WT mice were infected with βGal or Flag-HuR adenoviruses overnight.
Primary hepatocytes were also isolated from HuR flox/flox and HuR-HKO mice. Hepatocytes were treated with actinomycin D (10 mg/ml) for 0, 3, 6, and 12 h. Cells was harvested at indicated time, and RNA was isolated for RT-qPCR. The degradation rate of RNA (k) was estimated by plotting N t /N 0 against time and fitting to the following equation 32 : The half life of mRNA was calculated following this equation:

Statistical Analysis
Data were presented as means ± S.E. Differences between groups were analyzed by Student's t tests. p< 0.05 was considered statistically significant. *, p< 0.05. **, p< 0.01.

Cytosolic HuR is abnormally elevated in NASH livers.
Cytosolic translocation of HuR has been shown to regulate RNA stability 33,34 . To test whether the cytosolic translocation of HuR happens in NASH, we measured cytosolic, nuclear and total HuR protein levels in NASH by immunoblotting. As shown in Fig. 1a, b, cytosolic and total HuR protein levels were significantly increased in the livers of human patients with NASH, whereas the nuclear HuR protein levels were not altered in NASH (Fig. 1a, b). Consistently, in mice with MCD-induced NASH, cytosolic and total HuR protein levels were also dramatically increased, whereas the nuclear HuR protein levels were not altered (Fig. 1c, d). To further determine how HuR expression was increased in NASH, primary hepatocytes were isolated and treated with TNFα or palmitate acid (PA), as it is known that TNFα and PA could mimic inflammation and lipotoxicity in NASH, respectively 35,36 . As shown in Figure 1e-h, both TNFα and PA could significantly increase the HuR protein levels in both cytosol and total lysates, whereas the nuclear HuR levels were not changed under these two conditions. These data indicate that inflammation and lipotoxicity could increase both HuR expression and cytosolic localization of HuR, which may further promote NASH.

Hepatic deletion of HuR protects against MCD -induced NASH
To determine whether HuR regulates the NASH progression, we generated hepatocyte-specific HuR knockout (HKO) mice by cross HuR flox/flox mice with Alb-Cre transgenic mice. The genotype of HuR-HKO mice is HuR flox/flox Alb-Cre +/-. As expected, HuR protein levels were dramatically decreased by 96.2% in the livers of HuR-HKO mice (Fig. 2a). We did not observe any difference between HuR flox/flox and Alb-Cre mice (data not shown). HuR flox/flox mice also displayed similar MCD-induced NASH with Alb-Cre mice, as revealed by similar serum ALT activity ( Supplementary Fig. 1a), and similar liver TAG levels ( Supplementary Fig. 1b). Therefore, we used HuR flox/flox mice as the control for HuR-HKO mice in the following experiments. Under a normal chow diet feeding condition, the liver weights, liver TAG levels, and serum ALT activity were comparable between HuR-HKO and HuR flox/flox mice ( Fig. 2b-d). To further test whether HuR is involved in NASH progression, HuR-HKO and HuR flox/flox mice were fed with an MCD for 3 weeks. HuR-HKO mice protected against MCD-induced NASH, as revealed by lower serum ALT activities (Fig. 2e), lower liver weights and sizes ( Fig.   2f, g), less hepatic lipid droplets (Fig. 2h,i) and lower liver TAG levels ( Fig. 2j) compared with HuR flox/flox mice.
We then measured hepatocyte death by performing TUNEL assays in MCD feeding mice. The TUNEL-positive cells were significantly decreased in MCD feeding HuR-HKO mice (Fig. 2k). Death receptors (such as DR5 and FAS)/caspase8 signaling pathway has been shown to regulate hepatocyte death 8,37 . Downregulation of this signaling pathway may be associated with decreased hepatocyte death in HuR-HKO mice. To test this hypothesis, the expression of death receptors and the cleavage of caspase8 and caspase 3 were measured by immunoblotting. As shown in Fig. 2l, the protein levels of DR5 and the cleavage of caspase8 were much lower in the liver of HuR-HKO mice whereas the FAS protein levels were comparable between HuR-HKO and HuR flox/flox mice, which indicates that downregulation of DR5/caspase8 signaling pathway contributes to decreased hepatocyte death in HuR-HKO mice fed with MCD. Furthermore, liver fibrosis in MCD-fed HuR-HKO mice was ameliorated as revealed by less Sirius Red-positive areas in the liver sections than that in HuR flox/flox mice (Fig. 2m). These data suggest that hepatic deletion of HuR protects against MCD -induced NASH.

Hepatic deletion of HuR decreases expression of inflammation and cell death-related genes
To comprehensively compare the gene expression profiles in the livers of HuR-HKO and HuR flox/flox mice fed an MCD, we performed an RNA sequencing (RNA-seq) analysis. As shown in Fig. 3a, 353 genes were upregulated and 422 genes were downregulated. Gene Ontology (GO) analysis showed that genes related to leukocyte migration, leukocyte chemotaxis, cell chemotaxis, neutrophil migration, Fc receptor signaling pathway, and positive regulation of apoptotic process were significantly decreased (Fig. 3b), whereas those associated with renal system development, kidney development, urogenital system development and steroid biosynthetic process were upregulated ( Supplementary Fig. 2). qPCR analysis further confirmed these data. As shown in Fig. 3c, Mmp9, Tgf1β Fig. 3c, the expression of genes associated to VLDL secretion (ApoB and Mttp) and fatty acid β-oxidation (Ppara) were decreased in the livers of HuR-HKO mice. These data suggest that VLDL secretion and fatty acid β-oxidation less likely contributes to the decreased liver steaotosis in HuR-HKO mice. qPCR data also showed that genes associated to fatty acid uptake (Cd36 and Fatp5) and lipogenesis (Fasn, Dgat1, Scd1, and Chrebp) were reduced (Fig. 3c), which may contribute to the decreased liver steaotosis in HuR-HKO mice under an MCD feeding condition. However, we did not observe any difference in the expression of genes related to fatty acid uptake and lipogenesis between HuR-HKO and HuR flox/flox mice mice fed with a normal chow ( Supplementary   Fig. 3), which suggests that hepatic deletion of HuR does not primarily affect lipid metabolism. We consistently observed that DR5 was significantly downregulated in HuR-HKO mice under both normal chow and MCD feeding conditions (Fig. 2l), indicating that hepatic deletion of HuR primarily affects DR5 expression.

Hepatic overexpression of HuR induces hepatocyte death and liver injury
Next, we asked whether hepatic overxpression of HuR could induce hepatocyte death and liver injury. We generated liver-specific HuR-overexpressing (LOE) mice were generated by injecting purified HuR adenovirus.
Same amount of βGal adenovirus injection served as Control. As expected, Flag-HuR levels were increased in the livers of HuR-LOE mice (Fig. 4a). Liver-specific overexpression of HuR caused liver injury, as revealed by increased blood ALT activity (Fig. 4b), increased liver weight (Fig. 4c), decreased blood glucose (Fig. 4d), increased immune cell infiltration into the liver (Fig. 4e), and increased TUNEL-positive cells (Fig. 4f). The increased cell death was likely duo to DR5/caspase 8/caspase 3 signaling pathway in the livers of HuR-LOE mice (Fig. 4g). The expression of DR5 was significantly increased in HuR-LOE mice (Fig. 4g, h). These data indicate that hepatic overexpression of HuR leads to hepatocyte death and liver injury, which is likely due to increased DR5 expression.

HuR promotes cell death in primary hepatocytes by increasing both DR5 expression and the cleavage of caspase 8 and caspase 3
To determine whether HuR directly triggers hepatocyte death, we performed cell viability assay in HuR-overexpressing primary hepatocytes. The expression levels of Flag-tagged HuR were high (Fig. 5a), and the cell viability were significantly decreased (Fig. 5b). To comprehensively analyze the transcriptinal changes in the HuR-overexpressing primary hepatocytes, we performed an RNA sequencing (RNA-seq) analysis. As shown in Fig. 5c, 2677 genes were upregulated and 2712 genes were downregulated. Gene Ontology (GO) analysis showed that genes related to inflammation and apoptotic signaling pathway were significantly increased, whereas the genes related to catabolic process were dramatically decreased (Fig. 5d). qPCR analysis further confirmed these data. As shown in Fig. 5e To determine whether this cell death induced by HuR is associated with death receptors/caspase8/caspase 3 pathway, we measured death receptor protein levels and the cleavage of caspase 8 and caspase 3. As shown in Fig. 5f, the DR5 protein levels and the cleavage of caspase 8 and caspase 3 were significantly increased in HuR-overexpressing hepatocytes. We did not observe any difference in FAS protein levels between βGal and HuR groups (Fig. 5f), indicating that HuR does not regulate FAS expression. TRAIL, the ligand for DR5, was not regulated by HuR (Fig. 5e). These data demonstrate that HuR induces hepatocyte death by enhancing DR5/caspase8/caspase3 pathway.

HuR is essential for PA＆TNFα-induced cell death in primary hepatocytes
A combination of PA and TNFα (PA＆TNFα) can induce hepatocyte death that mimics NASH-associated hepatocyte death. To determine whether HuR is essential for PA＆TNFα-induced hepatocyte death, we isolated primary hepatocytes from HuR-HKO and HuR flox/flox mice and treated these cells with PA＆TNFα. As shown in Fig. 6a, HuR deficiency protected hepatocytes from PA＆TNFα-induced cell death. Expectedly, HuR and DR5 protein levels were increased by PA＆TNFα treatment in HuR flox/flox hepatocytes, whereas their protein levels were significantly decreased in HuR-HKO hepatocytes (Fig. 6b). Moreover, the cleavage of caspase 8 and caspase 3 was increased by PA＆TNFα treatment in HuR flox/flox hepatocytes, but their cleavage was significantly blocked in HuR-HKO hepatocytes (Fig. 6b). The ligand for DR5 is TRAIL. To determine whether HuR is essential for TRAIL-induced activation of caspase8 and caspase3, primary hepatocytes were isolated from HuR-HKO and HuR flox/flox mice, and TRAIL-induced cleavage of caspase8 and caspase3 was measured by immunoblotting. As shown in Fig. 6c, the cleavage of caspase 8 and caspase 3 were significantly decreased in HuR-HKO hepatocytes. Consistently, DR5 protein levels were significantly decreased in HuR-HKO hepatocytes (Fig. 6b, c). These data demonstrate that HuR is essential for PA＆TNFα-induced hepatocyte death and the cleavage of caspase8 and caspase 3, most likely due to the decreased DR5 protein levels.

HuR directly binds to the 3＇-UTR of DR5 transcript and decreases its mRNA decay.
HuR has been shown to binds to AREs in 3'-UTR and regulate mRNA stability. Photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) sequencing showed that HuR binds to DR5 transcript 40 . RNA immunoprecipitation (RIP) sequencing analysis also showed that HuR binds to DR5 (TNFRSF10B) mRNA 41 . Sequence analysis showed that 3'-UTR of DR5 mRNA contains several AREs, which further indicates that HuR may bind to 3'-UTR of DR5 mRNA and regulate DR5 mRNA stability. To test this hypothesis, we performed RIP-RT-qPCR analysis in both HuR-overexpressing and HuR knockout hepatocytes. The successful HuR precipitation by both Flag-beads and HuR antibody was confirmed by immunoblotting (Fig. 7a, b). As expected, both HuR and Flag immunoblotting signals were observed in signal of HuR in immunoprecipitation or immunoblotting was observed in HuR deficient hepatocytes (Fig. 7a,   b). RIP-RT-qPCR showed that a huge increase of DR5 mRNAs retrieved by Flag-beads in Flag-HuR-overexpressing hepatocytes and also observed a significant reduction of DR5 mRNA retrieved by HuR antibody in HuR-HKO hepatocytes (Fig. 7c, d). To examine the effect of HuR on DR5 mRNA stability in hepatocytes, we used Actinomycin D to inhibit mRNA transcription in HuR-HKO and HuR-overexpressing hepatocytes and measured the decay rate for DR5 mRNA. The half-life of DR5 mRNA was increased from 5.77 to 7.66 h in the HuR-overexpressing hepatocytes (Fig. 7e), while the half-life of DR5 mRNA was decreased from 7.22 to 5.14 h in the HuR-HKO hepatocytes (Fig. 7f). Taken together, HuR may induce hepatocyte death by binding and stabilizing DR5 mRNAs, leading to the activation of caspase8/caspase3 signaling pathway.

Discussion
Hepatocyte death is at the center of the second hits during NASH pathogenesis because it triggers liver inflammation, liver injury, and fibrosis. Clinical studies and genetic manipulations demonstrate that hepatocyte apoptosis plays a key role in NASH progression 8,9,10,11,12 . However, whether RNA processing regulates death signaling pathway during NASH progression remain poorly understood. In this study, we demonstrated that abnormal elevated HuR promotes hepatocyte apoptosis, inflammation and diet-induced NASH in mice by stabilizing DR5 mRNA. Hepatic deletion of HuR ameliorates MCD-induced NASH by decreasing DR5/caspase 8/caspase 3 signaling pathway, which provides a new therapeutic target for the treatment of NASH in humans.
Several lines of evidence support the pathological significance of HuR in controlling NASH progression.
Hepatic HuR, especially the cytosolic HuR, is abnormally upregulated in both human patients with NASH and mice with MCD-induced NASH. PA and TNFα could increased both HuR expression and cytosolic localization of HuR in primary hepatocytes, which may be partially due to PKC-mediated phosphorylation of HuR 33 .
Hepatic deletion of HuR ameliorates the severity of diet-induced NASH in mice, as shown by the reduced serum ALT activity, the decreased hepatic steatosis, the decreased hepatocyte death, and the reduced liver inflammation. In contrast, liver-specific overexpression of HuR induces hepatocyte death and liver injury. HuR 13 overexpression induced hepatocyte death in vitro in a cell-autonomous manner, demonstrating a direct role of HuR in triggering liver damage.
HuR regulates biological and pathophysiological processes by targeting and stabilizing ARE-enriched transcripts 42,43 . HuR overexpression in primary hepatocytes causes hepatocyte death by increasing ARE-enriched transcripts, which includes apoptosis and inflammation-related transcripts. One of the most robust response transcripts is DR5. DR5 activation directly triggers caspase 8 cleavage to induce its activation and further increases the cleavage of caspase 3, leading to apoptosis 37 . DR5 plays a key role in fatty acid-induced cell death and is also induced during NASH pathogenesis 9,10 . Knockdown of DR5 ameliorates fatty acid-induced hepatocyte death 9 . However, the pathophysiological cues that impinge on DR5 remain largely unknown. Our current work uncovers DR5 mRNA stability mediated by HuR as a key regulator of NASH progression. HuR directly binds to the 3＇-UTR of DR5 mRNA, and then strongly increases DR5 expression, which leads to the activation of caspase 8 and caspase 3 in hepatocytes, resulting in the increased hepatocyte death and liver injury. More importantly, HuR is essential for DR5 expression in the liver. Hepatic deletion of HuR decreases the expression of DR5 in the isolated primary hepatocytes and the livers of mice fed with either a normal chow diet or an MCD diet, which is most likely due to the decreased RNA stability of DR5 transcript. HuR deficiency ameliorates PA＆TNFα-induced hepatocyte death, which is likely due to decreased both DR5 expression and the activation of caspase8/caspase3 signaling pathway. These results demonstrate that HuR is essential for the activation of DR5/caspase8/caspase3 signaling pathway in hepatocytes.
Although some lipid metabolism-related genes were downregulated in HuR-HKO mice fed with an MCD, no big difference was observed in lipid metabolism between HuR-HKO and HuR flox/flox mice fed with a normal chow. Overexpression of HuR does not increase the expression of genes related to lipid metabolism. These results suggest that HuR does not primarily regulate lipid metabolism.
In conclusion, we have demonstrated that HuR is a novel important regulator of NASH progression. Our data also reveal a new mechanism in which HuR increases the mRNA stability of DR5. Abnormally elevated HuR expression leads to increased mRNA stability of DR5, which further increases hepatocyte death, 14 contributing to NASH progression. Our results also indicate that HuR may serve as a novel drug target for the treatment of NASH. (a) HuR protein levels in cytosol, nuclei, and total cell lysate from human NASH and normal liver tissues were measured by immunoblotting.

Figure Legends
(b) Quantification of the data in (a).
(c) HuR protein levels in cytosol, nuclei, and total cell lysate from the livers of MCD (3 weeks)-fed mice were measured by immunoblotting.
(d) Quantification of the data in (c).
(e) Primary hepatocytes were isolated from C57BL6 mice and treated with TNFα (40 ng/ml) for 2 hours.
HuR protein levels in cytosol, nuclei, and total cell lysate were measured by immunoblotting.
(f) Quantification of the data in (e).
(h) Primary hepatocytes were isolated and treated with palmitate acid (0.5 mM) for 18 hours. HuR protein levels in cytosol, nuclei, and total cell lysate were measured by immunoblotting.
(h) Quantification of the data in (g).
n=3-5 for each group. Each experiment was repeated at least twice with similar results.
(e)Representative H&E staining of liver sections from Control and HuR-LOE mice.
(g) The death receptors (FAS and DR5) /caspase8/caspase3 signaling pathway was measured by immunoblotting in Control and HuR-LOE mice.
(d) Top GO biological process terms enriched in upregulated and downregulated genes.
(a) Primary hepatocytes were isolated from HuR-HKO and HuR flox/flox mice and treated with PA (1 mM) and TNFα (100 ng/ml) for 28 hours. Cell viability assay was measured by MTT.
(b) Primary hepatocytes were isolated from HuR-HKO and HuR flox/flox mice and treated with PA (1 mM) and TNFα (100g/ml) for 24 hours. The cleavage of caspase8 and caspase3 was measured by immunoblotting.
(c) Primary hepatocytes were isolated from HuR-HKO and HuR flox/flox mice and treated with TRAIL (1μg/ml) for 2 hours. The cleavage of caspase8 and caspase3 was measured by immunoblotting.
These experiments were at least repeated twice with similar results. *, p< 0.05. **, p< 0.01. Data represent the mean ± SEM. (a,b) The successful HuR immunoprecipitation by both Flag-beads and HuR antibody, respectively, was confirmed by immunoblotting .
(c,d) RIP-RT-qPCR showed that a huge increase of DR5 3 ＇ -UTR retrieved by Flag-beads in Flag-HuR-overexpressing hepatocytes and that a significant reduction of DR5 3＇-UTR retrieved by HuR antibody also observed in HuR-HKO hepatocytes (n=3).