Figure 4. Inhibiting non-EGFR receptor tyrosine kinases increases dynamic Erk activity in keratinocytes.

(A) Representative traces from keratinocytes treated with either a Class 2 B-Raf inhibitor, a Class 3 RTK inhibitor, or DMSO as a control. Cells were imaged every 3 min over 12 h. (B-C) Average Erk pulse frequencies (in B) and overall Erk activity offset (in C) for keratinocytes treated as in A or with a neutralizing antibody targeting Met or VEGFR2. Each biological replicate (white circle) represents dynamics assessed from at least 100 single cells. Statistics are derived using a two-sided t-test (*, p<0.05; **, p < 0.01; ***, p < 0.001). (D-E) Western blot quantification for phospho-Erk (ppErk; in D) and phospho-EGFR (pEGFR; in E) in keratinocytes after the addition of either EGF or a non-EGFR RTK inhibitor (golvatinib or tivozanib). pEGFR and ppErk are normalized to total Erk protein and each value is represented as fold-induction relative to the untreated initial timepoint. (F) Representative single-cell traces showing Erk activity in keratinocytes that were pre-treated for 1 h with the EGFR inhibitor lapatinib or DMSO control, and then stimulated with EGF, Class 2 or 3 inhibitors, or additional DMSO. (G) The overall Erk activity offset a for cells treated as in F. Each biological replicate (white circle) represents the mean of at least 100 cells imaged for 8 h. (H) A conceptual model of these results whereby inhibition of non-EGFR receptor tyrosine kinases by small molecules or neutralizing antibodies acts downstream of EGFR to enhance EGFR-driven pulsatile Erk signaling