Figure 7. Silencing of Daxx expression in HepG2 cells by siRNA and potentiation of apoptosis by 4-HNE and CH11 antibody.

(A) Silencing of Daxx was performed by the ON-TARGET plus SMART pool Daxx siRNA as per the manufacturer’s instructions (Thermo Scientific Dharmacon) and control cells were treated with ON-TARGET plus Non-targeting siRNA in a similar way. After transfection, the cells were harvested after 48 h and the expression level of Daxx was examined by Western blot analysis. (B) Western blot analysis showing the enhanced activation of caspase-3 and JNK in Daxx depleted cells after 4-HNE treatment (20 µM HNE for 2 h). (C) Enhanced activation of apoptosis was also performed by In Situ immunofluorescence study. The cells (2 × 10⁵) were grown on glass cover slips in twelve-well plate and transfected with non targeting siRNA or Daxx siRNA. After 48 h of siRNA transfection, the cells were treated with either 50 µg/well Fas-agonistic antibodies (CH11) or 20 µM 4-HNE for 2 h at 37°C. The activation of caspase-3 in these cells was examined by CaspACE FITC-VAD-FMK in situ marker as per the manufacturer’s instructions and then observed under a fluorescence microscope.