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. 2019 Jul 17;10(8):542. doi: 10.1038/s41419-019-1761-4

Fig. 2. Exposure to propofol induces caspase-1-dependent pyroptosis.

Fig. 2

BMDMs were exposed to 0, 12, 60, 300 and 600 μM propofol. Following 3 h of propofol exposure, immunoblots of cell extracts and supernatants from BMDMs for proteins associated with pyroptosis (a) and apoptosis (b) were performed. c BMDMs were treated with propofol (300 μM) or vehicle for the indicated time periods. Immunoblots were performed to detect pyroptosis-related proteins in BMDM supernatants and cell extracts. d Immunoblots were performed to detect apoptosis-related proteins in cell extracts. GAPDH was the internal control. e BMDMs were treated with 300 μM propofol, and then, cell samples were harvested at 0, 10, 20, 30, 60, 120 and 180 min after propofol exposure. Cells were labelled with ICT’s poly-caspase inhibitor reagent. Active caspase-1 and active caspase-3/7 were analysed with a 96-well fluorescence plate reader (Spark 10 M, Tecan). f Following 6 h of propofol exposure, the levels of IL-1β were determined by ELISA in the supernatants of caspase-1-/- BMDMs. g Pre-treatment (30 min) of BMDMs with VX-765 or Z-VAD-FMK blocked 300 μM propofol-induced cytotoxicity at 3 and 6 h. However, when the concentration of propofol was increased to 600 μM, caspase-1 inhibitors did not significantly change the results of the CCK-8 or LDH release assays. h Pre-treatment (30 min) of BMDMs with VX-765 or Z-VAD-FMK inhibited high-dose propofol-induced cleaved caspase-1. Band intensity was quantified by ImageJ software, and the values of target protein were normalised to that of GAPDH. i Three-hundred micromolar propofol-induced pyroptosis was inhibited by treating cells with VX-765 and Z-VAD-fmk. Immunofluorescence staining with DAPI (blue), PI (red) and of cleaved caspase-1 (green) in BMDMs after 6 h of exposure. Representative microscopy images. Bars indicate a scale of 50 μm. The results are representative of three independent experiments. Bars represent the mean ± SE (n = 3–5); one-way ANOVA followed by the Duncan’s test; *P < 0.05 and **P < 0.01 versus control. CASP1 = caspase-1; CASP11 = caspase-11; CASP3 = caspase-3; CASP7 = caspase-7; CASP9 = caspase-9; Sup = supernatant; Cell ext = cell extract; WCL = whole-cell lysates