Figure 6: EGFR internalization and intracellular retention in a specific subset of MVBs is required for EGFR TK activation and delays onset of stress-induced apoptosis.

(a) Immunoblotting HeLa lysates showed transient, strong EGFR-T669 phosphorylation and gradually increased EGFR-Y1068 phosphorylation after UVC exposure, compared with weak T669 and rapid, strong Y1068 signal after EGF stimulation. (b) Immunoblotting HeLa lysates pre-incubated for 30 min with SB, exposed to UVC and incubated in the continuous presence of SB, showed that EGFR-Y1068 phosphorylation requires p38 activity. (c) Immunoblotting HeLa lysates showed that 30 min pre-incubation with dynasore prevented EGFR-Y1068 and ERK1/2 phosphorylation induced 1 h post UVC exposure, but not that induced by 30 min exposure to EGF. (d) Immunoblotting HeLa lysates showed that AP2α siRNA treatment inhibited EGFR-Y1068 phosphorylation up to 1 h post UVC exposure. (e, top) Immunoblotting PAE lysates showed that EGF-stimulated Y1068-phosphorylation is similar in PAE EGFR-ΔAP2 and -wt cells. However, although EGFR-wt showed increased Y1068-phosphorylation 15 min post UVC exposure, −ΔAP2 did not. Note that exposure time for p-EGFR Y1068 in EGF-stimulated samples has been reduced to avoid film saturation. (e, bottom) Quantification of EGFR-Y1068 phosphorylation from above. Data were normalized to control (untreated) EGFR-wt and are mean±s.e.m. of three independent experiments, *P<0.05 (Student’s t-test). (f) Immunoblotting HeLa lysates showed that 30 min SB treatment after UVC-induced EGFR internalization reduced EGFR-Y1068 phosphorylation that was not rescued by Rab11 siRNA treatment. (g) Immunoblotting HeLa lysates showed that ALIX siRNA treatment reduced EGFR-Y1068 phosphorylation and prevented ERK1/2 phosphorylation 1 h post UVC exposure. (h, left) AP2α RNAi results in increased percentage of TUNEL-positive HeLa cells 2 h post UVC exposure compared with control RNAi. (h, right) PAE EGFR-ΔAP2 showed increased TUNEL-positive cells 8 h post UVC exposure compared with EGFR-wt cells. Data are mean±s.e.m. of three independent experiments, *P<0.05 (Student’s t-test). (i) ALIX RNAi causes a similar increase compared with control RNAi to that caused by AP2α RNAi in the percentage of TUNEL-positive HeLa cells 2 h post UVC exposure. Data are mean±s.e.m. of three independent experiments, *P<0.05 (Student’s t-test).