Fig. 2
c-Src was activated through c-Src-Caspase-8. a, Immunoblotting analysis of phospho-Caspase-8 (pY380-Casp8) and phospho-c-Src (pY416-Src) in A549 + control/Casp8/c-Src shRNA cells plated on fibronectin (10 μg/ml) for the indicated amounts of time. b, Immunoblotting analysis of phospho-Caspase-8 and phospho-c-Src in A549 cells transfected with a control vector and H522 cells transfected with adenovirus encoding the control vector, wild-type Caspase-8 (Casp8 WT) or Y380A Caspase-8 (Casp8 Y380A) plated on fibronectin (10 μg/ml) for 1 day. c, A549 cells (upper panel) transfected with either control shRNA or Casp8 shRNA via lentivirus were plated on fibronectin (10 μg/ml) in the presence of a c-Src-specific fluorescent substrate, and fluorescence was recorded as a function of time (t test, *, P < 0.05). H522 cells (lower panel) transfected with a control vector, Casp8 WT, or Casp8 Y380A via adenovirus were plated on fibronectin (10 μg/ml) in the presence of a c-Src-specific fluorescent substrate, and fluorescence was recorded as a function of time (t test, *, P < 0.05). d, pY380-Casp8 and pY416-Src immunoblot analysis of A549 + control shRNA and H522 + control vector/Casp8 WT /Casp8 Y380A cell extracts immunoprecipitated with anti-Casp8 or anti-HA after tumor cells were allowed to attach to fibronectin (10 μg/ml) for 4 days. pY380-Casp8 immunoblot analysis was performed by a pulldown assay with 2 μg of GST-SH2 domains from c-Src and Abl; GST itself was used as a control. Anti-Casp8 and anti-c-Src immunoblotting of immunoprecipitated total cell lysates is shown at the bottom. e, Ectopically grown A549 (upper panel)/H522 (lower panel) tumors were surgically removed. Biweekly quantification of bioluminescence showed accelerated tumor growth and increased spontaneous metastasis in the mice implanted with A549 + control shRNA and H522 + Casp8 WT cells (t test, * P < 0.05). Data are presented as the mean ± SD. f, Total cumulative incidences of micrometastasis confirmed by immunostaining in the tumor-implanted mouse cohorts (χ2 test, *, P < 0.05). Micrometastatic events in various BALB/c nude mouse organs were confirmed by immunostaining. Tissue sections were scored as positive or negative based on the presence or absence of detectable micrometastasis