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. 2014 Oct 14;111(43):15438–15443. doi: 10.1073/pnas.1412767111

Fig. 3.

Fig. 3.

HSV-1 infection-induced necrosis requires MLKL but is independent of TNFR, TLR3, CYLD, and DAI. (A) MEFs isolated from WT, TNFR1 KO, TNFR1/TNFR2 double KO, and TLR3 KO mice were treated as indicated for 16–18 h. The cell survival rate was determined by measuring ATP levels. T, TNF-α; S, Smac mimetic; Z, z-VAD. (B) Total mRNA was collected from the indicated MEFs. The mRNA expression levels of the indicated genes were measured. (C) MEFs were transfected with NC or CYLD siRNA oligos for 48 h. Cells were treated as indicated for an additional 16–18 h. The cell-survival rate was determined by measuring ATP levels. Cell lysates were collected 48 h posttransfection and subjected to Western-blot analysis. (D) WT MEFs, RIP3 KO MEFs, or two DAI KO clones (32# and 40#) were infected with HSV-1 for 16–18 h. The cell-survival rate was determined by measuring ATP levels. Genomic DNA was collected from DAI KO clone 32# or 40#, and alterations of DAI DNA sequence were analyzed by PCR. (E) MEFs or MLKL-shRNA stable cell lines as indicated were infected with the HSV-1 F strain for 16–18 h. Cell lysates were collected and subjected to Western-blot analysis. The cell-survival rate was determined by measuring ATP levels. MLKL-shRNA, MEFs stably expressing a shRNA targeting mouse MLKL.