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. 2019 Mar 15;10(4):256. doi: 10.1038/s41419-019-1491-7

Fig. 5. Heat shock inhibits NLRP3 inflammasome activation.

Fig. 5

Cells were stimulated with LPS (100 ng/mL for BMDMs and 10 ng/mL for THP-1) for 20 h or not when indicated and then submitted or not to a heat shock (HS, 42 °C for 1 h followed by 2 h at 37 °C). a BMDMs from WT C57BL/6 mice were lysed and immunoblotted with indicated antibodies. b and c After ATP (5 mM, 30 min), nigericin (Nig, 40 µM, 30 min), Alum (100 µg/mL, 6 h) or MSU (100 µg/mL, 6 h) treatments, IL-1β was measured by ELISA (b) and caspase-1 p10 cleavage fragment was detected by western blot (c) in the supernatants of BMDMs. d THP-1 cell lysates were incubated at 37 °C or 4 °C, at indicated times. Caspase-1 p20 cleavage fragment was detected by western blot. Procaspase-1 was used as a loading control. e After ATP (5 mM, 30 min) or nigericin (Nig, 40 µM, 30 min) treatments, human IL-1β was measured with the HEK-Blue™ IL-1β cells and caspase-1 p20 cleavage fragment was detected by western blot in the supernatants of THP-1 cells. f After nigericin (Nig, 40 µM, 30 min) treatments, BMDMs were stained with ASC and NLRP3 antibodies and the proportion of cells with ASC/NLRP3 specks was evaluated. g Heat shocked BMDMs treated or not with nigericin (30 min, 10 µM) were stained with anti-NLRP3 and anti-HSP70 antibodies and assayed for PLA and the percentages of cells with fluorescent dots were evaluated. h and i BMDMs from HSP70−/− C57BL/6 mice were treated as in (b) and IL-1β was measured by ELISA in the supernatants (h) and the proportion of cells with ASC/NLRP3 specks was evaluated (i). Data are the mean of three independent experiments ± s.d. Statistics compare cells with or without heat shock: **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s. not significant using two tailed t test