Fig. 5

Overexpression of Nodal enhances susceptibility to apoptosis in OCCCa cells. a After treatment of the stable Nodal-expressing ES-NoD and mock cells with 20 μM CDDP for the times indicated, cells undergoing apoptosis (sub-G1) were detected by flow cytometry. This experiment was performed in triplicate using independent samples. b Left: after treatment with 20 μM CDDP, the stable Nodal-expressing ES-NoD and mock cells undergoing apoptosis (indicated by arrows) were detected by TUNEL assay. Original magnification, × 400. Right: number of apoptotic cells per 10 high power fields (HPFs) detected by TUNEL assay for the times shown. c Left: western blot analysis of the indicated proteins in the stable Nodal-expressing ES-NoD and mock cells after 20 μM CDDP treatment for the times shown. Right: values of endogenous Bcl2 relative to Bax protein were calculated by normalization to β-actin in the stable Nodal-expressing ES-NoD and mock cells after 20 μM CDDP treatment for the times shown. d Ishikawa cells were transfected with XIAP reporter constructs, together with cotransfection of Smad2 and/or Nodal. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means ± SDs. The experiment was performed in duplicate