HIV-1 Vpr mediated induction of IL-6 and IL-8 in astrocytes involves MAPK pathway.

2015-08-13T03:02:47Z (GMT) by Mohitkumar R. Gangwani Anil Kumar
<p>SVGA astrocytes were cultured and seeded in 6 well plates. The cells were 1h pre-treated with chemical inhibitor for MAPK pathway (SB203580 –p38; SP600125 –Jnk and UO126—Erk) and then transfected with a plasmid encoding HIV-1 Vpr or were mock-transfected. The cells were harvested at 6h post-transfection followed by the determination of IL-6 and IL-8 mRNA expression levels using real-time RT-PCR. The cell culture supernatants were collected at 48h post-transfection, and protein concentration of secreted IL-6 and IL-8 was determined using BioPlex multi-cytokine assay. <b>(A, C)</b> mRNA expression levels of IL-6 and IL-8 calculated relative to mock-transfected controls in the presence of chemical inhibitors, respectively. <b>(B, D)</b> Protein concentrations for secreted IL-6 and IL-8 with chemical inhibitors, respectively. <b>(E, F)</b> Depicts the effect of SB203580 and SP600125 on phosphorylated p38 and Jnk MAPK’s, respectively. <b>(G, H)</b> represents the effect of Erk-MAPK inhibitor UO126 on the mRNA expression levels of IL-6 and IL-8, respectively. <b>(I, K)</b> portray mRNA levels while <b>(J, L)</b> show protein concentration for IL-6 and IL-8 when individuals p38-MAPK isoforms were silenced, respectively. Every bar represents the mean ± SE of thee independent experiments done in triplicates. Statistical analyses were performed using 1-way ANOVA using post-hoc Tukey HSD test, <b>#</b> p < 0.01, <sup><b>a</b></sup> p < 0.05 as compared to Vpr transfected cells; <b>**</b> p < 0.01 and <b>*</b> p < 0.05 compared to mock-transfected controls.</p>