Figure 8. Sustained signaling from EGFR... | Rockefeller University Press

Figure 8.

$Sustained signaling from EGFR to ERK1/2 is dependent on the activity of the cell surface pool of EGFR and unaffected by the inhibition of endocytosis.(A) Serum-starved HeLa/Grb2-mNG cells were imaged by TIRF microscopy before and after stimulation with EGF (4 ng/ml) at 37°C. Selected time frames before EGF addition and after 5 min and 15 min of cell incubation with EGF are shown in the top row. Corresponding images of individual objects defined as the diffraction-limited puncta are shown in the bottom row. (B) Quantification of the number of Grb2-mNG puncta per FOV in time course sequences exemplified in A was performed as described in Materials and methods. Mean object numbers per FOV from the last six time points before EGF stimulation were calculated (EGF-independent background) and subtracted from the object numbers quantitated at each time point after EGF stimulation in corresponding FOVs. Mean values of background-subtracted object numbers per FOV (n = 3) with SDs are plotted against time. (C) Effect of EGFR inhibition by cetuximab (Ctxb). Serum-starved parental HeLa cells were stimulated with 1 ng/ml EGF for 5 or 15 min. The cells were either lysed or further incubated with EGF and with or without cetuximab (5 µg/ml) for an additional 15 min (15 min chase). Lysates were subjected to Western blotting with pMEK1/2, total MEK1/2, pERK1/2, total ERK1/2, and EGFR antibodies. (D and E) MEK1/2 (D) and ERK1/2 (E) activities were quantitated from experiments exemplified in C and expressed as the percentage of the maximum activity after 5 min of EGF stimulation. Bar graphs represent mean values (±SEM) obtained from four independent experiments. P values against cetuximab-untreated cells were calculated using an unpaired Student’s t test. (F) Parental HeLa cells were transfected with nontargeting (NT) or CHC targeted siRNAs. 3 d after transfection, the cells were stimulated with EGF (4 ng/ml) for the indicated times at 37°C. The lysates were probed by Western blotting with pERK1/2, CHC, ERK1/2, and EGFR antibodies. ERK1/2 activity was measured as described in E and expressed as the percentage of the maximum activity in NT-transfected cells. Mean values with SEMs (n = 3) are plotted against time. (G) Parental HeLa cells were pretreated with vehicle (DMSO) or Dyngo-4a (30 µM) for 30 min at 37°C and then stimulated with EGF (4 ng/ml) for the indicated times at 37°C. The aliquots of lysates were probed by Western blotting with pERK1/2, total ERK1/2, and EGFR antibodies. ERK1/2 activity was measured as described in E and expressed as the percentage of the maximum activity in vehicle-treated cells. Mean values with SEMs (n = 3) are plotted against time.$

Sustained signaling from EGFR to ERK1/2 is dependent on the activity of the cell surface pool of EGFR and unaffected by the inhibition of endocytosis. (A) Serum-starved HeLa/Grb2-mNG cells were imaged by TIRF microscopy before and after stimulation with EGF (4 ng/ml) at 37°C. Selected time frames before EGF addition and after 5 min and 15 min of cell incubation with EGF are shown in the top row. Corresponding images of individual objects defined as the diffraction-limited puncta are shown in the bottom row. (B) Quantification of the number of Grb2-mNG puncta per FOV in time course sequences exemplified in A was performed as described in Materials and methods. Mean object numbers per FOV from the last six time points before EGF stimulation were calculated (EGF-independent background) and subtracted from the object numbers quantitated at each time point after EGF stimulation in corresponding FOVs. Mean values of background-subtracted object numbers per FOV (n = 3) with SDs are plotted against time. (C) Effect of EGFR inhibition by cetuximab (Ctxb). Serum-starved parental HeLa cells were stimulated with 1 ng/ml EGF for 5 or 15 min. The cells were either lysed or further incubated with EGF and with or without cetuximab (5 µg/ml) for an additional 15 min (15 min chase). Lysates were subjected to Western blotting with pMEK1/2, total MEK1/2, pERK1/2, total ERK1/2, and EGFR antibodies. (D and E) MEK1/2 (D) and ERK1/2 (E) activities were quantitated from experiments exemplified in C and expressed as the percentage of the maximum activity after 5 min of EGF stimulation. Bar graphs represent mean values (±SEM) obtained from four independent experiments. P values against cetuximab-untreated cells were calculated using an unpaired Student’s t test. (F) Parental HeLa cells were transfected with nontargeting (NT) or CHC targeted siRNAs. 3 d after transfection, the cells were stimulated with EGF (4 ng/ml) for the indicated times at 37°C. The lysates were probed by Western blotting with pERK1/2, CHC, ERK1/2, and EGFR antibodies. ERK1/2 activity was measured as described in E and expressed as the percentage of the maximum activity in NT-transfected cells. Mean values with SEMs (n = 3) are plotted against time. (G) Parental HeLa cells were pretreated with vehicle (DMSO) or Dyngo-4a (30 µM) for 30 min at 37°C and then stimulated with EGF (4 ng/ml) for the indicated times at 37°C. The aliquots of lysates were probed by Western blotting with pERK1/2, total ERK1/2, and EGFR antibodies. ERK1/2 activity was measured as described in E and expressed as the percentage of the maximum activity in vehicle-treated cells. Mean values with SEMs (n = 3) are plotted against time.