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. 2014 Feb 11;24(4):417–432. doi: 10.1038/cr.2014.17

Figure 6.

Figure 6

Src kinase activity is required for TNF-induced necroptosis. (A) The Src protein level was analyzed in control and Src-knockout cell lines. The control and three Src-knockout cell lines were named as Control, Src-1#, Src-2# and Src-3#, respectively. The cells were challenged with TNF (10 ng/ml) and cell viability was measured. (B) Cells were pretreated with or without general tyrosine kinase inhibitor Genistein (100 μM) for 2 h or Src kinase family-specific inhibitor PP2 (2 μM) for 6 h. Cells were then challenged with TNF for 10 h. Cell viability was measured. (C) Parts of Control and Csk-knockdown cells were harvested and the cell extracts were subjected to western blot to examine the levels of Src and pTyr418-Src, and other parts of Control and Csk-knockdown cells were treated with TNF for 8 h. Cell viability was measured. (D) L929 cells were infected with control lentivirus or the indicated lentivirus expressing WT Src or constitutively active mutant Src-Y529F for 48 h. PI-positive dots (dead cells) were counted under a microscope. The average numbers of dead cells per 1 000 cells obtained from at least three different views of about 500 cells per view are shown. (E) Control or Gγ10-knockdown L929 cells were infected with lentivirus-expressing Vector, HA-Src (WT) or HA-Src-Y592F. A low dose of lentivirus was selected to adjust the expression of Src-Y529F to a modest level so that Src-Y529F-expressing cells did not undergo spontaneous necroptosis in the absence of TNF stimulation. A total of 48 h after lentivirus infection, cells were challenged with TNF for 10 h. Cell viability was measured. #P < 0.01, t test. (F) Peritoneal macrophages were treated with LPS (100 ng/ml) plus zVAD (20 μM) for different periods of time. Tyr418 phosphorylation level of Src was measured. (G) Peritoneal macrophages were pretreated with or without PP2 (2 μM) for 6 h and then challenged with LPS (100 ng/ml) plus zVAD (20 μM) for 20 h. PI-positive dots (dead cells) were counted under a microscope. The average numbers of dead cells per 1 000 cells obtained from at least three different views of about 500 cells per view are shown. Data in A-E, and G depict mean ± SEM of one representative experiment of three or more. See also Supplementary information, Figure S4.