HNSCC cell lines contain cell populations with low proteasome activity and higher sphere-forming capacity. HNSCC cell lines were engineered to express the fusion protein ZsGreen and the c-terminal of the degron ornithine decarboxylase (cODC). (a) Cell lines were cultured in log-growth phase in DMEM, and representative bright field and green fluorescent pictures of monolayer cells are shown. (b) The percentage of ZsGreen-cODCpos cells increases when HNSCC cells are cultured in serum-free media as tumorspheres. (c) Percentage of cells forming spheres from the ZsGreen-cODCneg (ZsG-) and ZsGreen-cODCpos (ZsG+) population after sorting by flow cytometry into 96-well plates. Means ± SD from four independent experiments are shown. (d) Representative CD44 staining of a HNSCC patient-derived tumor sample. Tumor cells show uniform membrane staining for CD44 (red). Nuclei are counterstained with DAPI (blue). (e) and (f) Flow cytometry analysis of ZsGreen accumulation (Y-axis) and ALDH1 expression (X-axis) in UM-SCC-6 and UM-SCC-12 cells). ZsGreen-cODCpos cells with low proteasome activity are a subpopulation of ALDH1-expressing cells with 19% of ZsGreen-cODCpos UM-SCC-6 cells and 41.5% of UM-SCC-12 cells positive for ALDH1.