Fig. 3

MTAs upregulate membrane TNF transcription through the JNK/c-Jun axis. a Effect of transcriptional inhibitor actinomycin D (ActD) on MTA-induced cell death in L929 cells. b RNA-sequencing analysis of Tnf and Jun gene expression patterns during MTA treatment. UCSC genome browser images depict calculated FPKM (fragments per kilobase of transcript per million mapped reads) values in RNA-sequencing data. Gene expression levels are provided in DATA SET  1. c Effect of Jun knockout on MTA-induced necroptosis in L929 cells. For complementation, wild-type c-Jun was expressed in the knockout cells and its expression was detected by immunoblotting. d qRT-PCR analysis of Tnf mRNA level in MTA-treated WT and Jun KO L929 cells. e Immunoblotting analysis of TNF in P100 fractions of NCZ-treated WT and Jun KO L929 cells. f A panel of MAPK and NF-κB inhibitors was tested for necroptosis inhibition effect on MTA-treated L929 cells. All inhibitors were pretreated for 2 h followed by NCZ challenge. i, inhibitor. g Immunoblotting analysis of the JNK/c-Jun activation in whole cell lysates of NCZ-treated L929 cells. h Immunoblotting analysis of TNF accumulation in membrane fraction (P100) in the presence of JNK inhibitor (JNKi, SP600125) for NCZ-treated L929 cells. i Immunoblotting analysis of TACE expression in whole cell lysates of NCZ-treated L929 cells. j, k Fluorimetric assay of measuring TACE activity in both cell lysate (left panel) and membrane fraction (right panel) of NCZ (j) or PTX (k) treated L929 cells. D, DMSO; NCZ, nocodazole; PTX, paclitaxel. Cell viability was determined by measuring ATP levels. The data are represented as mean ± SEM of duplicate wells (a, c, and f). Results are reported from one representative experiment. Experiments were repeated independently for four (f), three (a, c, d, and g), or two (e and h–k) times