Figure 10. Induction of caspase-1 mediated cell death in HIV ssRNA40 treated microglial cells.

Activation of caspase-1 in ssRNA40 treated HMG. (a) Representative flow cytometric analysis for active caspase-1 mediated pyroptotic cell death in HMG cells at 24h post ssRNA40 treatment. (b) Measurement of pyroptotic cell death in HMG cells following HIV ssRNA40 mediated NLRP3 inflammasome by Annexin V and propidium iodide (PI) staining using flow cytometric analysis. Data are representative of three independent donors (n=3, *P<0.05). (c and d) Quantitative measurement of rate of pyroptotic cell death in microglia cells following their exposure to HIV ssRNA40. Quantitative measurement of active caspase-1 (p20) expression in culture supernatants (c) and nucleasomal DNA in the cytoplasmic fraction (d) derived from HMG cells stimulated with ssRNA40 for 24h and 48h by ELISA. Results are presented as mean ± SD, n=3; *P<0.05. (e) Pharmacologic inhibition of NLRP3 by MCCP950 inhibits ssRNA40 induced pyroptosis. HMG cells were pre-treated with NLRP3 inflammasome inhibitor, MCC950 (1μM and 10μM) or vehicle control prior to their exposure to ssRNA40 (5μg/mL) for 48h. Cells were analyzed for active caspase-1 by flow cytometry and culture supernatants were analyzed for cell death by LDH release assay. Top, representative histogram (left) and geometric mean intensity measurement (right) from flow cytometric analysis for intracellular active caspase-1 expression. Bottom, quantitative measurement of LDH release in the culture supernatants. Results are presented as mean ± SD, n=3; *P<0.05.