Fig. 6: IFNβ expression increases in LPS/NIG-stimulated macrophages and IFNβ promotes pyroptosis of ASCs.

a Heat map from RNA-seq analysis for ASCs exposed to SC3, SLN3, SCB, and SLNB respectively (n = 3). The heat map showed the expression level of 15 IFN-stimulated genes significantly up-regulated in SLNB-treated ASCs compared to SLN3-treated ASCs. Red (highest) to green (lowest) represent the values of gene expression level. b–e 3T3 cells, BMDMs, WT iBMDM, and Gsdmd^−/− iBMDM were incubating in LPS (1 μg/ml) for 5 h before being challenging with nigericin (10 μM) for 1 h. Then Cells were collected and evaluated for expression of IFNβ and IFNγ at mRNA levels by RT-PCR. Values were showed as the relative ratio of mRNA of IFNβ or IFNγ to mRNA of GAPDH. n = 3. f–h ASCs were pretreated with IFNβ (0.4 ng/ml) and LPS (0.5 μg/ml) for 4 h before being stimulated with LPS/NIG, SCB, or SLNB for 2 h. And then collected ASCs for immunoblot analysis (out of at least three performed) of GSDMD and IL-1β (e) or replaced with fresh medium for another 2 h. Harvest the medium for detecting LDH (f) and mature IL-1β (g) released by ASCs. n = 3. The statistical significance of the differences between various treatments was determined by a two-tailed t test for two groups or one-way analysis for three or more groups. Data are shown as mean ± SD. ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. IFN interferon, LPS lipopolysaccharide, NIG nigericin, ASCs adipose tissue-derived mesenchymal stem cells, BMDMs bone marrow-derived macrophages, iBMDMs immortalized bone marrow-derived macrophages, WT wildtype, GSDMD gasdermin D, GSDMD-NT N-terminal gasdermin D, SCB supernatant collected from BMDMs, SLNB supernatant collected from LPS/NIG-treated BMDMs, RT-PCR real-time reverse transcriptase-polymerase chain reaction, LDH lactate dehydrogenase.