Fig 3.

Hrk knockdown by siRNA was associated with significantly reduced wtE2F-1- or E2Ftr-induced apoptosis. SK-MEL-2 and A375 cells were infected with control vector, Ad-wtE2F-1 or Ad-E2Ftr at MOI 100, followed by transfection with control siRNA or Hrk siRNA as indicated. (A) RT-PCR was performed after 24 hrs of transfection as described in Materials and methods. The bar graph is expressed as Hrk fold increase relative to control vector-infected cells adjusted for α-actin as an internal control. Each experiment is a representation of three independent experiments performed in duplicate (Ctrl: control; bars: mean ± S.D.; *P < 0.05; **P < 0.01 compared with either wtE2F-1 or E2Ftr infection plus control siRNA transfection; n = 3). After 48 hrs of transfection, the percentage of cells showing typical apoptotic nuclei was counted by Hoechst 33258 staining under a fluorescence microscope (B) and determined by annexin V-PE staining by flow cytometry analysis (C). (D) After 48 hrs of transfection, caspase-9 activity was determined as in Figure 1F. Values represent mean ± S.D. of three independent experiments. (Ctrl: control; bars: mean ± S.D.; *P < 0.05; **P < 0.01 compared with either wtE2F-1 or E2Ftr infection plus control siRNA transfection; n = 3).