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. Author manuscript; available in PMC: 2019 Jun 27.
Published in final edited form as: Mol Cell. 2019 Apr 2;74(4):801–815.e6. doi: 10.1016/j.molcel.2019.02.038

Figure 4. IL-1β-Induced mtDNA Release Is NF-κB Dependent.

Figure 4.

(A) A549 was treated with media (0) or 10 ng/mL IL-1β for 3 h ± 1 h pretreatment with DMSO or 10 μM IKKα/β inhibitor (2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile [ACHP] from Bristol-Meyers Squibb [BMS]) before fractionation and protein analysis by immunoblot.

(B) A549 was treated as in (A) with 500 nM IKKβ inhibitor (PF184 and TPCA1).

(C) A549 was treated as in (A) with 1 μM TBK1/IKKε inhibitor (BX795).

(D) A549 was treated with media or 10 ng/mL IL-1β for 3 h ± 1 h pretreatment with DMSO or the indicated inhibitors before protein analysis by immunoblot.

(E) Total DNA was harvested from cytosolic and nuclear fractions of A549 treated as in (A)–(C) and analyzed by qPCR. Cytosolic mtDNA genes were normalized to respective nuclear RPL13A and presented as fold enrichment over media-treated controls. Statistical analysis was performed using two-way ANOVA with Bonferroni’s to compare each treatment to mock; n = 3 with mean ± SEM.

(F) A549 was treated with media or 10 ng/mL TNF-α for 3 h before fractionation and protein analysis by immunoblot.

(G) Total DNA was harvested from cytosolic and nuclear fractions of A549 treated as in (F) and analyzed by qPCR. Statistical analysis was performed using Student’s t test and Holm-Sidak to compare treatments; n = 3 with mean ± SEM. **p < 0.01; ***p < 0.001.