Characterisation of EGFR expression CHIR99021 GSK-3 inhibitor in the SCCHN cell panel The dependence of reovirus oncolysis on upregulated Ras signalling has been reported previously. Since selleck chem Calcitriol Ras signalling can be driven by EGFR Inhibitors,Modulators,Libraries stimulation and SCCHN overexpresses EGFR, the panel of cell lines was evaluated for EGFR expression levels with a view to neither Inhibitors,Modulators,Libraries assessing whether reovirus Inhibitors,Modulators,Libraries sensitivity could be predicted by measuring EGFR expression. FACS analysis of EGFR expression was carried out for the whole panel and 9 representative cell Inhibitors,Modulators,Libraries lines were also profiled for total and phospho EGFR by western blot. A broad range of cell surface EGFR levels was evident across the panel.
Similarly, total and phospho EGFR protein levels were also widely distributed Inhibitors,Modulators,Libraries in the cell lines tested.
HN5 and Cal27 expressed the highest amounts of EGFR by FACS and western blot.
Conversely, Inhibitors,Modulators,Libraries HN3 and SIHN 5B have relatively low Inhibitors,Modulators,Libraries levels of surface EGFR. Levels of total and phospho EGFR for SIHN 5B were undetectable by western blot, while HN3 had constitutively phosphorylated EGFR. Following profiling, the cell Inhibitors,Modulators,Libraries lines were ranked according to their EGFR ex pression by FACS and western analysis for either total or phospho EGFR resulting in 3 different ranks. To determine whether the FACS data correlate with total and/or phospho EGFR, the ranked data were plotted against each other. FACS data vs total EGFR western blot showed a strong positive correlation.
No correlation was evident between FACS analysis and phospho EGFR western blot, reveal ing that surface level EGFR analysis represents Inhibitors,Modulators,Libraries the levels of total EGFR protein in each Inhibitors,Modulators,Libraries cell line, rather Inhibitors,Modulators,Libraries than the ac tive signalling component.
Correlation between EGFR expression, Inhibitors,Modulators,Libraries GTP loading Inhibitors,Modulators,Libraries on Ras and reovirus sensitivity To test whether EGFR pathway activity, Inhibitors,Modulators,Libraries and, hence, sig nalling in the Ras pathway, was predictive inhibitor Tofacitinib of increased sensitivity to reovirus, the EGFR ranks obtained in Figure 3A and B were plotted against the ranks of reo virus IC50 dilution derived for the cell line panel. Total EGFR assessed either by FACS or western blot did not correlate with reovirus IC50 dilution.
Interestingly, U0126 buy a non statistically significant inverse correlation was seen between phospho EGFR and reovirus Inhibitors,Modulators,Libraries IC50 dilution.
The baseline GTP loading status of Ras was deter mined for 12 representative cell lines. The resulting western blot and densitometry data demonstrate that most cell lines had similar levels of Ras activation. Exceptions to this finding included SIHN 013, PJ41 and PJ34 cell lines. There was no significant more correlation between Ras activation status and sensitivity to reovirus. For further experiments, 4 cell lines were selected from the panel as being representative of the broad range of EGFR expression/reovirus sensitivity HN5 . HN3 . and SIHN 5B.