Erlotinib induces EGFR protein accumulation through protein stabilization. (A) Calu-3 cells were treated for the indicated period of time with 0.5 μM erlotinib. At the end of drug treatments cell lysates were immunoblotted to detect EGFR protein. (B) Calu-3 cells were treated with 0.5 μM erlotinib for 24 h then the drug was removed and drug-free medium was added for further 24 and 48 h. Then, cell lysates were immunoblotted to detect the EGFR protein levels. (C) Calu-3 cells were treated for 24 h with erlotinib 0.5 μM, in the absence/presence of 0.1 μg/ml actynomicin D and 2 μg/ml cyclohexymide. At the end of the experiment, cell lysates were immunoblotted to detect the indicated proteins. The immunoreactive spots were quantified by densitometric analysis, ratios of EGFR/Actin were calculated and values are expressed as fold increase versus control. (D) Calu-3 cells were exposed to 0.5 μM erlotinib for the indicated period of time and the EGFR mRNA was detected by RT-PCR. The mean values of two independent measurements (± SD) are shown. (E) EGFR, p-P70S6K, p-P44/42 and P44/42 were detected by Western blotting in Calu-3 cells untreated or treated for 24 h with 1 μM gefitinib, erlotinib and lapatinib, 10 μg/ml cetuximab, 10 μM U0126, 1 μM NVP-BKM-120 and NVP-BYL-719 and 100 nM RAD001. The results are from representative experiments. Each experiment, repeated three times, yielded similar results.