Figure 7.

Time-lapse monitoring of γ-H2AX formation in U2OS cells transduced with 3F4 Fabs. Nonlabeled (A) or Alexa Fluor 488-labeled (B) 3F4 Fabs were delivered by electroporation to U2OS cells. 24 h post-transduction, the cells were treated with either HU (A) or G+V (B). After 48 h of HU treatment for 48 h or G+V treatment for 24 h, the cells were fixed and analyzed by immunofluorescence microscopy. The pictures show typical fields of the transduced cells observed under the microscope. The non-labeled Fabs were revealed with Alexa Fluor 488-labeled goat anti-mouse immunoglobulin (AF 488-GAM) and the Alexa Fluor 488-labeled Fabs (AF 488-Fab) were visualized without an additional processing. The nuclei were counterstained with DAPI. (C) Analysis of the cell fate by time-lapse microscopy following transduction. After transduction (TR), the U2OS cells were either nontreated (upper panel) or treated with G+V (lower panel) as indicated and subjected to time-lapse monitoring. Phase-contrast images of several fields were taken every 30 min during approximately 8 h. The micrographs show representative individual cells of a minimum of 50 cells from three independent experiments recorded by fluorescence and phase-contrast at the beginning of the time-lapse analysis (TLA) and, subsequently, by phase-contrast. Numerous cell division events within the population of the nontreated transduced cells were visible at approximately 30 h post-transduction (upper panel). In the presence of G+V, the cells did not divide and some rounded up and became floating after approximately 50 h of incubation (lower panel). Magnification: 400×.