Figure 2. Hypoxia down-regulates global membrane protein endocytosis.

(a) HeLa cells were pre-treated at normoxia or hypoxia (1% O₂) for 20 h, followed by cell-surface biotinylation, staining with streptavidin-AF-488 and visualization by confocal microscopy. Scale bar, 20 μm. (b) FACS quantification of biotinylated cell-surface proteome in HeLa cells shows inhibition by hypoxic treatment for the indicated time periods. (c) Immunoblotting for biotinylated cell-surface proteins from a similar experiment as described in (b) shows down-regulation by hypoxia. (d) Confocal microscopy imaging of endocytosed, biotinylated membrane proteins following 30 min of internalization and cell-surface biotinylation depletion in HeLa cells shows inhibition by hypoxic treatment for the indicated time periods. Scale bar, 10 μm. (e) FACS quantification of the endocytosed membrane proteome from a similar experiment as described in (d) shows down-regulation by hypoxia. (f) Immunoblotting for endocytosed proteins from a similar experiment as described in (d) shows inhibition by hypoxia. (g–l) FACS quantification of biotinylated cell-surface proteome (g–i) and endocytosed membrane proteome (j–l) following 30 min of internalization performed with the indicated cell types (A549, lung adenocarcinoma; MDA-MB-231, breast adenocarcinoma; U87-MG, glioblastoma) treated at normoxia or hypoxia for 20 h. Data are presented as % relative to normoxic cells (in b and g–i) or as % of total cell-surface biotinylation at normoxia and hypoxia, respectively, at t=0 (in e, and j–l)±s.d. from three independent experiments. *P<0.05 (Student's t-test).