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. 2017 Dec 4;216(12):4107–4121. doi: 10.1083/jcb.201701107

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© 2017 Hos et al.

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Figure 3. — p62 levels contribute to IFN-I–mediated cell death upon S. Typhimurium infection. (A and B) Immunofluorescence staining of p62. After infection with S. Typhimurium, WT and Ifnar1^−/− BMDMs were immunostained for p62 (A), and the number of p62-positive puncta per cell was quantified (B). n = 50 cells each, repeated three times. Bars, 10 µm. (C) Relative Sqstm1 mRNA expression. WT and Ifnar1^−/− BMDMs were infected with S. Typhimurium for the indicated time, and Sqstm1 (the gene encoding p62) mRNA expression was determined by real-time PCR. Values were normalized to the amounts of mRNA in uninfected BMDMs. Two independent experiments with three replicates each. (D and E) Immunoblot analysis of autophagic flux. WT and Ifnar1^−/− BMDMs were either infected with S. Typhimurium or pretreated with Concanamycin A (ConcA; 100 nM, 2 h) and then infected with S. Typhimurium in the presence of 50 nM Concanamycin A for the indicated time. Samples were immunoblotted for p62 (D), and p62 expression from three independent experiments was quantified densitometrically (E). Concanamycin A inhibits lysosomal acidification of the autophagosome and is used to determine autophagic flux. ST, S. Typhimurium. UI, uninfected. (F) Immunoblot analysis of p62 knockdown efficiency. WT and Ifnar1^−/− BMDMs were transfected with nontargeting (siCtrl) or siRNA specific for Sqstm1/p62 (sip62). Thereafter, total cell lysates were analyzed for p62 by Western blotting. (G) Luminescence analysis of p62-dependent cell death. Luminescence signals of WT and Ifnar1^−/− BMDMs transfected with nontargeting (siCtrl) or Sqstm1/p62-specific (sip62) siRNA were assessed to determine cell viability 6 h after S. Typhimurium infection. Values were normalized to the luminescence signal of uninfected WT BMDMs. Results are representative of three independent experiments performed in quintuplicates and are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.s., not significant.