Fig. 5

TCP-1/TNFα combined with TCP-1/IFNγ induced late apoptosis/secondary necrosis. a Hochest and PI double staining of tumor frozen sections from control and TCP-1/TNFα and TCP-1/IFNγ combined treatment groups. PI is only permeant to dead cells. PI positive staining was found in tumor cells in the TCP-1/TNFα and TCP-1/IFNγ combination treatment group but not control group indicating combination treatment induced late apoptosis/secondary necrosis. b H & E staining of tumor frozen sections. The combination treatment group had condensed (pyknosis) and fragmented nucleus (karyorrhexis) suggesting the cells were undergoing late apoptosis or necrosis. c Western blot result of cleaved caspase-3 and cleaved PARP in the tumors. Semi-quantification of cleaved caspase-3 and PARP is done using ImageJ software. TCP-1/TNFα and TCP-1/IFNγ single treatment induced apoptosis as shown by induction of cleaved caspase-3 and PARP while the combined treatment group did not show such effect. d MTT and LDH assay of colon 26 cells treated by PBS, TNFα, IFNγ or TNFα plus IFNγ. Ratio of TNFα to IFNγ was determined according to in vivo study. TNFα and IFNγ synergistically inhibited colon 26 cell growth and caused membrane damage at 48 h. e Western blot result of cleaved caspase-3 and cleaved PARP in colon 26 cells treated for different time. Semi-quantification of cleaved caspase-3 and PARP is done using ImageJ software. TNFα plus IFNγ induced apoptosis earlier than TNFα or IFNγ alone. f Flow cytometry result of Annexin V and PI double staining. TNFα plus IFNγ induced massive late apoptosis/necrosis at 48 h. g Time course study was performed with TNFα plus IFNγ at different time points. Results demonstrated that this cell death was through early apoptosis to late apoptosis/necrosis. C control, T TNFα, I IFNγ, TT TCP-1/TNFα, TI TCP-1/IFNγ. Data were presented as mean ± SEM. *P < 0.05. ***P < 0.001