Radiation enriches for cells with low proteasome activity and increases self-renewal capacity. (a) HNSCC cells stably expressing the ZsGreen-cODC fusion protein were grown as monolayers cultures and treated with 5 daily fractions of 3 Gy. The number of ZsGreen-cODCpos cells was assessed 72 hours after the last fraction of radiation using flow cytometry. Shown are mean percentages of ZsGreen-cODCpos cells with standard deviation (SD). (b) Treatment of UM-SCC-6-ZsGreen-cODC and UM-SCC-12-ZsGreen-cODC sphere cultures were treated with 5 fractions of 3 Gy, also resulting in enrichment of ZsGreen-cODCpos cells with low proteasome activity. This effect was more pronounced in radiosensitive  UM-SCC-6 cells than in radioresistant  UM-SCC12 cells. (c and d) 72 hours after the last fraction of radiation, cells were plated in 96-well plates at clonal densities to assess self-renewal capacity. Mean (± SD) percentages of cells forming a sphere with are shown. (e) Representative FACS analysis of HNSCC cell lines (monolayer) after treatment with 0 or 5×3 Gy.