Role of p38MAPK, caspase-2 and caspase-8 on abrin-induced mitochondrial membrane potential (MMP) loss in Jurkat cells.

<p>(A) Jurkat cells treated with abrin alone or along with inhibitors were harvested and stained with DiOC6 dye and the percentage of cells positive for green fluorescence were analyzed by flow cytometry as described in Materials and methods. The blue line indicates untreated Jurkat cells and the black line represents Jurkat cells treated as indicated in each panel. Also, percentage in each panel denotes the loss of mitochondrial membrane potential.</p

Abrin mediated protein synthesis inhibition and apoptosis in Jurkat cells.

<p>(A) Jurkat cells were treated with different concentrations of abrin for 8 h and protein synthesis was measured by incorporation of [³H]-leucine. (B) Jurkat cells were treated with varying concentrations (16 nM – 0.016 nM) of abrin for 12 h. After the treatments cells were harvested, fixed with 70% ethanol, stained with propidium iodide and quantified for apoptotic population. Each bar is presented as mean ± SE of triplicate samples.</p

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Abrin activates DNA damage signaling pathway.

<p>Phosphorylation of H2AX (γH2AX) was analysed in Jurkat cells (A) treated with abrin (10 ng/ml) for different time intervals (0–10 h) (B) pretreated with broad spectrum caspase inhibitor, z-VAD.fmk for 2 h and then with abrin or (C) with gamma radiation (20 Gy). Equal protein loading was checked by stripping and re-probing the membranes for β-actin.</p

Abrin causes activation of p38 MAPK pathway which leads to apoptosis.

<p>Jurkat cells were pretreated for 2(10 ng/ml) for 10 h. (A&B) After the treatments, Cells were harvested fixed with 70% ethanol, stained with propidium iodide and quantified for apoptotic population. Each bar is presented as mean ± SE of triplicate samples. After the similar treatments,whole cell lysates were prepared and analysed by Western blot for (C) p-p38 MAPK, total p38 MAPK caspase-2, 8 and 3. (D) For p-JNK, total JNK and caspase-3. Equal protein loading was checked by stripping and re-probing the membranes for β-actin.</p

Abrin induces ER stress in Jurkat cells.

<p>(A) After the treatment of Jurkat cells with abrin (10 ng/ml) for different time intervals (0–10 h), whole cell lysates were prepared and analysed by Western blot for the total and phosphorylated levels of eIF2α, JNK and p38 MAPK using specific antibodies (B) Jurkat cells were pretreated with 50 μM z-VAD.fmk for 2 h followed by abrin (10 ng/ml) for 10 h. After the treatments, whole cell lysates were prepared and analysed by Western blot for the total and phosphorylated level of eIF2α, JNK and p38 MAPK using specific antibodies.</p

Chikungunya Virus Exploits miR-146a to Regulate NF-κB Pathway in Human Synovial Fibroblasts

<div><p>Objectives</p><p>Chikungunya virus causes chronic infection with manifestations of joint pain. Human synovial fibroblasts get infected with CHIKV and could lead to pro-inflammatory responses. MicroRNAs have potentials to regulate the gene expression of various anti-viral and pro-inflammatory genes. The study aims to investigate the role of miR-146a in modulation of inflammatory responses of human synovial fibroblasts by Chikungunya virus.</p><p>Methods</p><p>To study the role of miR-146a in CHIKV pathogenesis in human synovial cells and underlying inflammatory manifestations, we performed CHIKV infection in primary human synovial fibroblasts. Western blotting, real-time PCR, luciferase reporter assay, overexpression and knockdown of cellular miR-146a strategies have been employed to validate the role of miR-146a in regulation of pro-inflammatory NF-κB pathway.</p><p>Results</p><p>CHIKV infection induced the expression of cellular miR-146a, which resulted into down-regulation of TRAF6, IRAK1, IRAK2 and increased replication of CHIKV in human synovial fibroblasts. Exogenous expression of miR-146a in human synovial fibroblasts led to decreased expression of TRAF6, IRAK1, IRAK2 and decreased replication of CHIKV. Inhibition of cellular miR-146a by anti-miR-146a restored the expression levels of TRAF6, IRAK1 and IRAK2. Downregulation of TRAF6, IRAK1 and IRAK2 led to downstream decreased NF-κB activation through negative feedback loop.</p><p>Conclusion</p><p>This study demonstrated the mechanism of exploitation of cellular miR-146a by CHIKV in modulating the host antiviral immune response in primary human synovial fibroblasts.</p></div

List of Primers.

<p>List of Primers.</p

MicroRNA sequences.

<p>MicroRNA sequences.</p

Overexpression and knockdown of miR-146a influences CHIKV replication.

<p>miR-146a enhances replication of CHIKV. 24 h post-transfection of scramble miR-146 and miR-146a (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103624#pone-0103624-t001" target="_blank">Table-1</a>), synovial cells were infected with CHIKV at MOI of 2 and harvested 32 h post infection for RNA isolation. CHIKV transcript levels were quantified via qPCR by using primers against distal 500 bp of 3′UTR region of CHIKV genome, normalized with cellular GAPDH transcript level. (<b>A</b>) Graph representing mRNA levels of CHIKV in miR-146a transfected cells as compared to scrambled miR-146a, normalized with GAPDH mRNA levels. (<b>B</b>) Graph representing the mRNA levels of CHIKV in cells transfected with anti-miR-146a compared with cy3 labeled controls, normalized with GAPDH transcript level. Three biologically independent experiments were performed and data is presented as mean ± SEM. *p<0.05.</p