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. 2019 Feb 21;10:268. doi: 10.3389/fimmu.2019.00268

Figure 2.

Figure 2

ERS-induced mitochondrial dysfunction is involved in inflammasome activation. (A) Immunoblot analysis at different time-points of NLRP3 and pro-IL-1β in lysates of BMDMs infected with M. bovis (MOI 10). (B) Immunoblot analysis of NLRP3 and pro-IL-1β in lysates from BMDMs infected for 24 h with M. bovis (MOI 10) in the presence or absence of 4-PBA. Control image is the same as Figure 1D. (C) ROS production measured at various time-points by flow cytometry in BMDMs infected with M. bovis (MOI 10). (D) ROS production was measured by flow cytometry in BMDMs infected for 48 h with M. bovis (MOI 10) in the presence or absence of 4-PBA. (E) qPCR analysis of mtDNA release into the cytosol during M. bovis (MOI 10) infection. (F) Immunoblot analysis at different time-points of cytochrome c in cytosolic extracts of BMDMs infected with M. bovis (MOI 10). (G) qPCR analysis of mtDNA release into the cytosol in BMDMs treated with or without 4-PBA for 1 h and then infected with M. bovis (MOI 10) for 48 h. (H) Immunoblot analysis of cytochrome c in cytosolic extracts from BMDMs infected with M. bovis (MOI 10) for 48 h in the absence or presence of 4-PBA. (I) IL-1β immunoblot analysis of supernatants from BMDMs infected for 24 h with M. bovis (MOI 10) in the presence or absence of CsA. (J) ELISA for IL-1β detection in supernatants from BMDMs treated with or without CsA for 1 h and then infected with M. bovis (MOI 10) for 24 h. Control image is the same to Figure 1E. ELISA for (K) TNF-α and (L) IL-6 quantification in supernatants from BMDMs treated with or without CsA for 1 h and then infected with M. bovis (MOI 10) for 24 h. Control image is the same to Figures 1H,I. LPS+ATP, positive control for inflammasome activation, 200 ng/ml, and 1 mM, respectively; LPS, positive control for TNF-α and IL-6 production, 200 ng/ml; 4-PBA, 4-phenyl butyric acid, ERS inhibitor, 5 mM; UNT, untreated; rotenone, positive control for ROS production, 40 μM; TM, tunicamycin, positive control for ERS-induced mitochondrial damage, 10 μg/mL; CsA, cyclosporine A, inhibitor of MPTP opening, 10 μM; siNLRP3, silencing RNA for NLRP3, 50 nM; siAIM2, silencing RNA for AIM2, 50 nM; siCon, non-targeting control siRNA, 50 nM. MOI, multiplicity of infection. For (C,E), data are representative of at least three independent experiments. The results are shown as the mean ± SD. The asterisks indicate statistically significant differences compared with untreated cells (**P < 0.01, ***P < 0.001, n.s., not significant). P-values were analyzed by using one-way ANOVA followed by post-hoc Tukey's test. For (D,G,J–L), data are representative of at least three independent experiments, each performed in triplicate for WB. The results are shown as mean ± SD. **P < 0.01, ***P < 0.001, n.s., not significant. P-values were analyzed by using Student's t-test.