Figure 1: Dual role of p38 in ligand-independent stress-triggered EGFR trafficking in HeLa cells.

(a) EGFR immunolocalization in untreated versus UVC-exposed cells 1 h post UVC exposure (left), and quantification of surface downregulation in cells exposed to UVC or treated with EGF for the indicated times (right). Arrows indicate perinuclear accumulation of EGFR (green) after UVC exposure. Data are mean±s.e.m. of three independent experiments. (b) Untreated or UVC-exposed HeLa cells were fixed after 1 h, or further incubated with the p38 inhibitor SB202190 (SB) for 1 h (left). Untreated or EGF-treated cells were fixed after 30 min, or further incubated with SB for 1 h in the continuous presence of EGF (right). p38 inhibition causes EGFR (green) redistribution to the plasma membrane following UVC, but not EGF exposure. (c) Pre-treatment for 30 min with PITSTOPII prevents UVC-induced EGFR (green) internalization (left) but PITSTOPII addition 1 h after UVC-induced EGFR internalization does not affect perinuclear EGFR accumulation or the recycling induced by simultaneous p38 inhibition (right). (d) Cells transfected with control or Rab11 siRNA were immunoblotted after 72 h for Rab11 and tubulin to assess knockdown efficiency (top). Rab11 knockdown did not prevent UVC-induced EGFR (green) internalization but prevented EGFR recycling after subsequent SB treatment (bottom). (e) Cells transfected with EGFR-GFP were fixed 1 h after UVC exposure and ultrathin cryosections were immuno-labelled for EGFR with 8 nm gold. EGFR-GFP (arrows) is on the limiting membrane and ILVs of MVBs. (f) Immunofluorescence analysis of UVC and EGF sequentially exposed HeLa cells (see Methods for experimental details). Red arrows show endosomes containing EGFR (green) and EGF (red). White arrows show EGFR+ve, EGF-ve endosomes, indicating a separate subset of MVBs containing stress-internalized but not EGF-bound EGFR. Scale bar, 5 μm. (g) Cells transfected with constitutively active Rab5-Q79L-DsRed were exposed to UVC and incubated for 1 h before treatment with EGF-AlexaFluor 647 (red) for 3 h. Red and white arrows show EGFR+ve/EGF+ve and EGFR+ve/EGF-ve endosomes, respectively. Scale bars, 10 μm for confocal and 100 nm for EM, unless otherwise indicated; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.