Fig. 2: Supernatant collected from LPS/NIG-treated BMDMs induces pyroptosis of ASCs.

The table showed the different supernatants collected from BMDMs or 3T3 cells. a LPS-primed ASCs were treated with supernatants of 3T3 cells and BMDMs for 2 h. After treating with related supernatants, the cells were collected and lysed for immunoblot analysis or replaced the supernatant with fresh media for another 2 h. At the end of experiment, the medium was harvested for detecting LDH, mature IL-1β, and caspase-1 p20 release of ASCs. Immunoblot analysis of caspase-11, proCaspase-1, GSDMD, and IL-1β in cell lysates of ASCs. b–d immunoblot analysis of caspase-11, proCaspase-1, and GSDMD, IL-1β in cell lysates of ASCs after treating with indicated supernatant. Data are representative of at least three independent experiments. LPS-primed ASCs were treated with 1 μg/ml LPS plus 10 μM nigericin or the indicated supernatants of 3T3 cells and BMDMs for 2 h. After the above treatments, collected the cells for immunoblot analysis or replaced the supernatant with fresh media for another 2 h. e Representative microscopic photographs of ASCs treated with SC3, SCB, SLN3, and SLNB respectively. As stated in a, photographs were taken by at 2 h after treatment. Objective magnification: ×40, bar 25 μm. Red arrows indicated vacuoles; black arrows indicated cell swelling. f–h As stated in a, the medium was harvested for detecting LDH (f), mature IL-1β (g), and caspase-1 p20 (h) release levels of ASCs. Data are representative of at least three independent experiments. n = 3. Statistical significance was determined using one-way analysis of variance. Data are shown as mean ± SD. ns: p > 0.05, ****p < 0.0001. LPS lipopolysaccharide, NIG nigericin, ASCs adipose tissue-derived mesenchymal stem cells, BMDMs Bone-marrow-derived macrophages, SN supernatant, GSDMD gasdermin D, GSDMD-NT N-terminal gasdermin D, LDH lactate dehydrogenase.