Figure 5. Influence of 1,25D3 on the response to fractionated radiation in MCF-7/ATG5− cells. (A) ATG5− cells (top panel) or MCF-7 vector control cells (bottom panel) were exposed to radiation alone (5×2 Gy), or 1,25D3 prior to irradiation and viable cell number was determined by exclusion of trypan blue at the indicated days following the initiation of radiation exposure. ↑ Indicates when irradiation occurred. **p < 0.0001 from IR on corresponding day, ┼p < 0.0001 comparing corresponding time points in MCF-7/ATG5− cells and vector control cells. (B) Apoptosis was monitored by the TUNEL assay and DAPI staining 3 d post-irradiation. (C) Autophagic flux was based on the decline in p62 levels monitored by western blotting 24 h post-irradation. Actin was utilized as a loading control. Serum starvation was used as a positive control for autophagic flux. The bar graph presents densitometry data for the p62/β actin protein ratio normalized to controls.