detection-mitochondria-membrane-potential-to-study-clic4-knockdown

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis <em>In Vitro</em>

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

Anhui medical university

Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is a newly identified intracellular antiviral factor. By depleting the dNTPs pool of host cells to a low level that cannot support the efficient synthesis of viral cDNA, it restricts replication of some retroviruses. As a DNA virus, Hepatitis B virus (HBV) experiences a process of reverse transcription in its life cycle akin to that of retroviruses. However, whether SAMHD1 can restrict HBV replication in liver cells is unknown. Here, we reported that SAMHD1 expression was detectable in four liver cell lines. Exogenous expression of SAMHD1 in SMMC-7721 cells restrained HBV replication. Similarly, SAMHD1 impeded HBV replication in another liver cell line, BEL-7402. Remarkably, the catalytically inactive mutant, SAMHD1 HD/AA also hampered HBV replication. Additionally, HBV replication reduced SAMHD1 expression in HepG2 cells. Moreover, it was found that IFN-α induced expression of SAMHD1 in liver cells. Together, these findings suggested that IFN-α-inducible SAMHD1 inhibited HBV replication in liver cells. 

Inhibition of Hepatitis B virus replication by SAMHD1.

Sterile α motif and HD domain-containing protein 1 (SAMHD1) is a novel intrinsic restriction factor that inhibits the replication of certain retroviruses and DNA viruses through its deoxynucleoside triphosphate triphosphohydrolase activity. A previous study by our group showed that SAMHD1 restrained hepatitis B virus replication and interferon (IFN)‑α induced SAMHD1 expression in liver cells. However the mechanisms of SAMHD1 upregulation by IFN‑α in liver cells have remained elusive. The present study demonstrated that IFN‑α treatment increased SAMHD1 mRNA levels in SMMC‑7721 cells in a time‑dependent manner. Knockdown of STAT1 inhibited the induction of SAMHD1 expression by IFN‑α in SMMC‑7721 cells. STAT2 silencing also suppressed the induction of SAMHD1 expression by IFN‑α in SMMC‑7721 cells. Furthermore, the induction of SAMHD1 expression in SMMC‑7721 cells by IFN‑α was found to be dependent on IFN‑regulatory factor 9 (IRF9). In conclusion, these results suggested that the interferon‑stimulated gene factor 3 complex, which consists of STAT1, STAT2 and IRF9, is required for the induction of SAMHD1 expression by IFN-α in SMMC-7721 cells.

Interferon-stimulated gene factor 3 complex is required for the induction of sterile α motif and HD domain-containing protein 1 expression by interferon-α in SMMC-7721 cells.

Human head and neck squamous carcinoma is the 6th most prevalent carcinoma worldwide. Although many novel therapies have been developed, the clinical treatment for patients remains non-ideal. Chloride intracellular channel 4 (CLIC4), one of the seven members of the CLIC family, is a newly found Cl(-) channel that participates in various biological processes, including cellular apoptosis and differentiation. Accumulating evidence has revealed the significant role of CLIC4 in regulating the apoptosis of different cancer cells. Here, we investigated the functional role of CLIC4 in the apoptosis of HN4 cells, a human head and neck squamous carcinoma cell line.

Knockdown of CLIC4 enhances ATP-induced HN4 cell apoptosis through mitochondrial and endoplasmic reticulum pathways.

In rheumatoid arthritis patients, the fibroblast-like synovial cells (FLS) growth is not controlled normally, but is similar to the tumor cells proliferation in histology. Our previous studies have shown that resveratrol inhibits the proliferation of FLS and promotes FLS apoptosis. However, the molecular mechanisms involved in resveratrol-induced FLS apoptosis have not been determined yet. Here, we showed that the FLS cell viability (following pretreatment with 5 µM H O for 24 hr) exhibited better proliferation performance than at other concentrations via the CCK-8 assay. The cell apoptotic rate increased with the increasing concentration of resveratrol (0, 40, 80, 160, 320 μM), as detected by TdT-mediated dUTP nick-end labeling (TUNEL) staining and western blotting. Furthermore, the expression level of autophagy-related proteins (LC3A/B, ATG-5) decreased with the increased concentration of resveratrol, as determined by immunofluorescence and western blot analysis. We also showed that resveratrol induced FLS mitochondrial morphology change. Moreover, mitochondrial function detection showed that the mitochondrial membrane potential was lost with the increased concentration of resveratrol as examined by the JC-1 assay. The production of ATP in cells was positively and negatively correlated with the resveratrol concentration. Simultaneously, the intracellular calcium release and calcium influx decreased gradually with the increase in resveratrol concentration. Therefore, we proposed that resveratrol can reduce the level of autophagy in FLS. The decrease in the autophagy level can lead to the accumulation of reactive oxygen species, which may result in mitochondrial dysfunction and promotion of FLS apoptosis. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Article	Year
Inhibition of Hepatitis B virus replication by SAMHD1. Biochemical and biophysical research communications\| PubMed ID: 25019997	2014
Interferon-stimulated gene factor 3 complex is required for the induction of sterile α motif and HD domain-containing protein 1 expression by interferon-α in SMMC-7721 cells. Molecular medicine reports\| PubMed ID: 26397446	2015
Knockdown of CLIC4 enhances ATP-induced HN4 cell apoptosis through mitochondrial and endoplasmic reticulum pathways. Cell & bioscience\| PubMed ID: 26816615	2016
Reducing-Autophagy Derived Mitochondrial Dysfunction during Resveratrol Promotes Fibroblast-Like Synovial Cell Apoptosis. Anatomical record (Hoboken, N.J. : 2007)\| PubMed ID: 29461680	2018

Jinsen Lu

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