Fig. 1

Hypoxia induces ligand-independent constitutive EGFR phosphorylation via inhibition of its endocytosis and lysosomal degradation. a Hypoxia-induced chronic but constitutive EGFR phosphorylation on a series of tyrosine residues. b pEGFR endocytosis rate in hypoxic HN4, CAL-27, HN6, and HN30 cells (n = 3). c Flow cytometric analysis of membrane-localized EGFR on hypoxic HN4, CAL-27, HN6, and HN30 cells (n = 3). Percentage of cells with high EGFR intensity was indicated. d The colocalization of pEGFR with the lysosome marker LAMP1 in hypoxic HN4 and CAL-27 cells, as shown by confocal microscopy images. The colocalization of pEGFR (red) and LAMP1 (green) was estimated with plot profile using ImageJ. Correlation coefficient R > 0.6 was considered well-collocated, while R < 0.6 was taken as poorly collocated. Bar, 20 µm. e Representative immunoblot images and half-life analysis of pEGFR and total EGFR levels of HN4 and CAL-27 cells after CHX treatment for the indicated time periods. Data were from representative results of at least three independent experiments. Data are represented as mean ± SD. ****P < 0.000 1; one-way ANOVA (c); Pearson’s R correlation (d)