Fig. 5

Treatment with the EGFR kinase inhibitor AG1478 arrests cells in G1 and leads to accumulation of RanBP17. A Treatment of UM-SCC-3 and UT-SCC-26A HNSCC cell lines with the EGFR kinase inhibitor AG1478 reduced cell proliferation (PCNA) but displayed an unexpected rise of RanBP17 protein expression levels (arrow, antibody orb226830). B Cell cycle sorting of UM-SCC-3 cells reveals a significant rise of RanBP17 and drop of PCNA in G1/G0 cells after AG1478 treatment. C Cell cycle analysis shows a reduction of cells in the S and elevation of cells in the G1/G0 phase of the cell cycle after AG1478 exposure (significant in UM-SCC-3) consistent with a block in the G1 phase as previously reported for this kinase inhibitor [21]. D A FACS based BrdU proliferation assay was performed for more detailed S-phase analysis. The S-phase was separated into three subcompartments (S1-S3, gates were adjusted manually for each sample to account for shifts in the whole cell population). A significant drop of cells in the early S phase (S1) was observed in both tested HNSCC cell lines. Cell numbers in S2 and S3 were significantly reduced in UM-SCC-3 but not UT-SCC-26A cells. Statistical tests: C (column chart) and D (left column chart): Unpaired Students t-test, two-sided; D (right column chart): One way ANOVA with Tukey´s correction for multiple comparisons. (n=4 for all analyses) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. TP53 mutation in UM-SCC-3: p.R248Q (nuclear p53); TP53 mutation in UT-SCC-26A: p.Y236* (cytoplasmic p53)