Figure 4

Ephrin B3 siRNA in combination with IR induces an increase in cell-death signaling and alleviates IR-induced G2/M arrest. U-1810 cells were transfected with non-target or Ephrin B3 siRNA for 48 h and then either irradiated (8 Gy) or left untreated before harvesting at the indicated time points. (a) Western blot analysis of PARP cleavage was performed 48 and 120 h post IR and GAPDH was used as a loading control. Results shown are one representative out of three experiments. (b) For analysis of nuclear morphology, cells were fixed in 70% EtOH and stained with Hoechst 33342. Left: cells displaying apoptotic morphology or mitotic catastrophe were quantified in 100 cells in three independent experiments using fluorescence microscopy. Bars represent S.D. Right: pictures showing the nuclear morphology of non-target or Ephrin B3 siRNA-transfected cells, with or without irradiation (8 Gy). (c) Cell cycle analysis was performed using staining of DNA with propidium iodide in which cells with hypodiploid DNA content (subG1 cells) were excluded from the analysis. In quantification only gated cell population was used. The cell cycle distribution after non-target siRNA or Ephrin B3 siRNA either alone or combined with IR (8 Gy) was analyzed at 48 h post IR. Left: flow cytometric histograms showing cell cycle distribution after transfection with Ephrin B3 or non-targeting siRNA in combination with IR. Right: quantification of the number of cells in individual cell cycle phases. Data presented are one representative experiment of three independent experiments