Fig. 4. HSP70 inhibits NLRP3 inflammasome through direct interaction with NLRP3.

a Cell lysates from LPS stimulated THP-1 cells (10 ng/mL) were supplemented with recombinant HSP70 and incubated at 37 °C (+) or left at 4 °C (−). Caspase-1 p20 cleavage fragment was detected by western blot. β-Actin was used as a loading control. b–c BMDMs from WT C57BL/6 mice were transfected with control vector (HA) or with HA-HSP70, stimulated with 100 ng/mL LPS for 20 h and then left untreated or treated 30 min with ATP (5 mM) or nigericin (Nig, 40 µM). b Cell lysates were immunoblotted to detect HSP70 expression (inset). β-Actin was used as a loading control. IL-1β was measured by ELISA and caspase-1 p10 cleavage fragment was detected by western blot in the supernatants. c Cells were stained with ASC and NLRP3 antibodies and the proportion of cells with ASC/NLRP3 specks vas evaluated. n.d.: not detected. d LPS primed THP-1 cells treated or not with nigericin (30 min, 10 µM) were lysed, immunoprecipitated with anti-HSP70 antibody and analysed by western blot. Neg: lysates without anti-HSP70. Ext: whole cell extracts. Numbers represent NLRP3 expression normalized to immunoprecipitated HSP70 (fold increase), mean of two independent experiments. e LPS primed BMDMs treated or not with nigericin (30 min, 10 µM) were stained with anti-NLRP3 and anti-HSP70 antibodies and assayed for PLA. Representative images are shown and the percentages of cells with fluorescent dots were evaluated. Data are the mean of at least three independent experiments ± s.d. Statistics compare HSP70 overexpressing BMDMs with HA transfected cells (b and c) or untreated with treated BMDMs (e): *p < 0.05; **p < 0.01 using two tailed t test