Expression of the <i>EGFR</i>-GS and—DEL mutant proteins enhances MCF10CA1a EGF-independent growth but not sensitivity to gefitinib.

<p><b>A.</b> Cells were cultured in the presence (+ EGF) or absence (- EGF) of EGF and their proliferation assessed using real-time imaging on the Incucyte Zoom and measuring the increase in confluency over 4–5 days. The doubling times of the cell lines were calculated from the rates of proliferation from 36–60 hours (24 hour period). While there was no difference in proliferation between the cell lines in the presence of EGF, in the absence of EGF all three <i>EGFR</i> constructs reduced MCF10CA1a doubling time (*p<0.05, **** p<0.0001^,, unpaired T-test, n = 6). <b>B.</b> Representative growth curves for cells grown in EGF-free media with measurements of confluency taken every 6 hours for up to 102 hours. <b>C-D.</b> Calculated IC₅₀ values of gefitinib for inhibition of proliferation (<b>C</b>) or cell toxicity (<b>D</b>) from cells treated for 48 hours in various doses of gefitinib and monitored in real-time using the Incucyte Zoom (** p<0.01, unpaired T-test, n = 3). <b>E</b>-<b>G.</b> Calculated mean IC₅₀ values from cells treated for seven days in various doses of gefitinib (<b>E</b>, n = 2), afatinib (<b>F</b>, n = 4) or cetuximab (<b>G</b>, n = 1) and cell death determined using the MTS assay. CA: MCF10CA1a-EV, WT: MCF10CA1a-<i>EGFR</i>-WT, GS: MCF10CA1a-<i>EGFR</i>-GS, DEL: MCF10CA1a-<i>EGFR</i>-DEL.