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

Figure 4.

Figure 4

NLRP3 and caspase-8 are required for ERS-induced mitochondrial damage. (A) qPCR analysis of mtDNA release into the cytosol in BMDMs treated with no inhibitor and belnacasan for 1 h and then infected with M. bovis (MOI 10) for 48 h. (B) Immunoblot analysis of cytochrome c in the cytosolic extracts from BMDMs treated with no inhibitor or belnacasan for 1 h and then infected with M. bovis (MOI 10) for 48 h. (C) Immunoblot analysis at different time points of caspase-8 in lysates of BMDMs infected with M. bovis (MOI 10). (D) Immunoblot analysis of caspase-8 in lysates of BMDMs treated with no inhibitor or 4-PBA for 1 h and then infected with M. bovis (MOI 10) for 48 h. (E) Immunoblot analysis of caspase-8 in lysates of BMDMs transfected with control non-targeting siRNA (siCon) or NLRP3 targeting siRNA (siNLRP3) and then infected for 48 h with M. bovis (MOI 10). (F) Immunoblot analysis of caspase-8 in lysates of BMDMs transfected with siCon or siAIM2 and then infected for 48 h with M. bovis (MOI 10). (G) Immunoblot analysis at different time points of cleaved caspase-8 in the mitochondrial fraction of BMDMs infected with M. bovis (MOI 10). (H) Immunoblot analysis of caspase-8 in the mitochondrial fraction of BMDMs infected with M. bovis (MOI 10) for 48 h in the presence or absence of 4-PBA. (I) Immunoblot analysis of caspase-8 in the mitochondrial fraction of BMDMs transfected with control non-targeting siRNA (siCon) or NLRP3 (siNLRP3) targeting siRNA and then infected for 48 h with M. bovis (MOI 10). (J) Immunoblot analysis of caspase-8 in the mitochondrial fraction of BMDMs transfected with control non-targeting siRNA (siCon) or AIM2 targeting siRNA (siAIM2) and then infected for 48 h with M. bovis (MOI 10). (K) qPCR analysis of mtDNA release into the cytosol in BMDMs treated with no inhibitor or z-IETD-fmk for 1 h and then infected with M. bovis (MOI 10) for 48 h. (L) Immunoblot analysis of cytochrome c in cytosolic extracts from BMDMs infected with M. bovis (MOI 10) in the absence or presence of z-IETD-fmk. UNT, untreated; belnacasan, inhibitor of caspase-1, 20 μM; TM, tunicamycin, positive control for ERS-induced caspase-8 cleavage, 10 μg/mL; 4-PBA, 4-phenyl butyric acid, ERS inhibitor, 5 mM; z-IETD-fmk, caspase-8 inhibitor, 50 μM; Fl casp8, full-length caspase-8; Cl casp8, cleaved caspase-8; 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. The data are representative of at least three independent experiments, each measured in triplicate. The results are shown as the mean ± SD. **P < 0.01, n.s., not significant. P-values were analyzed by using Student's t-test.