Figure 5

TIFA-mediated apoptosis depends on caspase activation, but not p53 accumulation. (a) Western blot was performed to analysis p53 activation in TIFA or TIFAΔ6 cells using specific anti-p53 and anti-phospho-p53 antibody. β-Actin was used as a loading control. (b) The bar graph shows the result of three separate experiments examining percentage of cell death after p53 suppression. Immunoblot detects cleaved caspase-3 expression in p53 suppressed cells using specific active/mature anti-caspase-3. β-Actin was included as a loading control. (c) 5-Bromo-2-deoxyuridine (BrdU)-FITC and 7-aminoactinomycin D (7-AAD) double staining was used to analysis cell cycle with p53 silence down cells. Representative statistical result was shown. The corresponding result of ki-67 expression was measured by western blot. (d) Immunoblotting of shCon cells (left panel) or shP53 cells (right panel) showing the expression of p53 and p21 in cell culture (Grow), upon the addition of Doxarubicin in plain media (1 ug) for 24 h (Dox), or control plain media (Starve). (e) The bar graph shows the statistics result of three separate experiments regarding percentage of cell death of SK-Hep1 TIFA cells treated with Z-VAD. Immunoblotting shows mature and cleaved forms of caspase-8 as well as cleaved caspase-3. β-Actin was included as a loading control. (f) BrdU-FITC and 7-AAD double staining was used to detect cell cycle after cell treated with Z-VAD. Representative statistics result was shown. The corresponding result of ki-67 expression was measured by western blot. (g) Possible model of TIFA in HCC. Reconstituting TIFA expression in HCC cell lines promoted two independent effects; the activation of caspases (3 and 8) possibly analogous to activation via classic complex 2 (TRADD/TRAF2) by TNFa or TLRs, as well as the induction of p53 and cell cycle arrest.