Reducing cytochrome c enhances NLRP3 inflammasome activation while exogenous cytochrome c inhibits the caspase-1 activation in a reconstitution assay.

<p>(A) Immunoblots of cell supernatants and cell lysates from LPS primed THP-1 previously transfected with cytochrome c or control siRNAs. The indicated proteins were immunoblotted. (B) Quantification of the results from (A) and 2 other similar experiments. Results are shown as mean +/- SEM of the fold increase of the indicated protein as assessed by Image J. The difference between the control and cytochrome c siRNA treated cells were compared by Student <i>t</i> test using Prism software. ** indicates that p < 0.01. (C) Immunoblot to assess caspase-1 activation <i>in vitro</i> using a cell fraction enriched for mitochondria and another for cytosolic proteins. The mitochondrial fraction was prepared from HEK 293T cells that had been treated with or without ATP. The cytosolic fraction was prepared from LPS primed THP-1 cells. The two fractions were mixed and incubated with either BSA or cytochrome c. The levels of the indicate proteins in the various mixtures are shown. (D) Quantification of the results from (C) and 2 other similar experiments. Results are shown as mean +/- SEM. The intensity of the bands determined using Image J. The differences in processed caspase-1 (p20) levels in the reactions containing BSA or cytochrome c were compared by Student <i>t</i> test using Prism software. ** indicates that p < 0.01. The above experiments were respectively performed 3 times.