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. 2017 Mar 1;12(3):e0172575. doi: 10.1371/journal.pone.0172575

Fig 6. NLRP3 inflammasome activation is prevented by blocking the caspase 3 –dependent release of MVs.

Fig 6

A. Western blot analysis of caspase-3 in total extracts of HepG2 cells not transfected (C), transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. Equal loading was confirm by re- probing the same membrane with monoclonal antibody against the house-keeping β-actin. B. Protein quantification of preparation of MVs as obtained from control HepG2 cells (C), from HepG2 cells transfected with the non silencing RNA and then exposed to PA (NsC) and HepG2 cells silenced for caspase 3 and then exposed to PA (siCASP3). Data are expressed as μg/μl and as mean ± SD being (n = 12 for any condition). C. Flow cytometric analysis of internalization of PKH26-fluorescent die-MVs in HepG2 naïve cells. Color legend: i) black trace is related to HepG2 naïve cells treated with ultracentrifugated solution (also marked with PKH26-fluorescent die) derived from cells just exposed to BSA (used as control), ii) pink trace is related to HepG2 naïve cells treated with MVs derived from HepG2 cells transfected with non-silencing treated with palmitic acid 0,25mM for 24hrs; iii) green trace is related to HepG2 naïve cells treated with MVs derived from HepG2 cells transfected with specific siRNA for caspase-3 and treated with palmitic acid 0,25mM for 24hrs. D. Western blot analysis of NF-Kb/p65 protein levels in cytosolic and nuclear extract obtained from HepG2 naïve cells exposed or not (C) to MVs derived from HepG2 cells transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. Equal loading was confirmed by re-probing membrane with α-tubulin and Lamin-A (cytosolic and nuclear house-keeping, respectively). E. Western blot analysis of NLRP3 protein levels as well as of cleaved caspase-1 (p-10-CASP-1) and IL-1β in total extracts obtained by HepG2 naïve cells exposed or not (C) to MVs derived from HepG2 cells transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. LPS 1mg/ml was used as positive control. Equal loading was confirmed by re-probing membrane with β-actin.