Figure 2: Hypoxia promotes endosome hyperacidification.
(a and b) Representative confocal images of pH-sensitive probe HPTS localization in (a) MDA-MB-231 or (b) HT-1080 cells labelled with Alexa Fluor 546-conjugated Tfn (endosomes) or Lysotracker (lysosomes). (c and d) Percentage of colocalization of HPTS with Alexa Fluor 546-conjugated Tfn or Lysotracker showing predominant labelling within endosomes in (c) MDA-MB-231 or (d) HT-1080 cells (n=3 independent experiments with >150 cells per experimental condition). Scale bar, 10 μm and original magnification is × 40. (e and f) Endosomal–lysosomal pH measured in (e) MDA-MB-231 or (f) HT-1080 cells cultured in normoxia or hypoxia (n=3 independent experiments with >75 cells per condition). (g and h) Endosomal pH (Tfn-positive vesicles) in (g) MDA-MB-231 or (h) HT1080 cells cultured for 4 h under normoxic or hypoxic conditions (n=3 independent experiments with >75 cells per experimental condition). (i and j) pHc measured with SNARF-1 pH-sensing probe in (i) MDA-MB-231 or (j) HT1080 cells cultured for 4 h under normoxic or hypoxic conditions in the presence or absence of EIPA, a selective NHE1 inhibitor (25 μM) (n=3 independent experiments with >75 cells per experimental condition). (k and l) Endosomal–lysosomal pH in (k) MDA-MB-231 or (l) HT-1080 cells cultured in normoxia or hypoxia for 4 h in the presence or absence of the neutralizing agents, Baf (100 nM) or Cq (10 μM) (n=3–5 independent experiments with >75 cells per experimental condition). Bars represent the mean±s.e.m. (*P<0.05, **P<0.01, ***P<0.001, unpaired Student’s t-test).
