Figure 5.

Chloroquine enlarges endosomes and induces co-localization of autophagy-related protein with endolysosomal markers. (A) SUM159PT cells were grown on cover glasses and treated with vehicle or 10 µM CQ. After 48 h, cells were fixed with cold methanol and incubated with anti-LC3B and anti-CD63 antibodies followed by corresponding secondary antibodies for confocal microscopy. Scale bars: 50 µm. (B) Endosome diameter was measured for n = 150 endosomes from control and CQ-treated cells, where significance was determined by two-tailed t-test (****: p < 0.001). (C) Pearson’s correlation coefficient (PCC) between CD63 and LC3B was determined for n = 11 individual cells treated with vehicle or CQ, where significance was calculated by two-tailed t-test (****: p < 0.001). (D) – (F) MDA-MB-231 cells stably expressing CD63-GFP were grown on cover glasses in full media. After 48 h of 10 µM CQ treatment, cells were fixed and immunostained as indicated for confocal microscopy. To determine co-localization, the intensity profile along a line in a single z-section (as indicated in the boxed region) was measure for each channel (except DAPI) and compared, where overlap was interpreted as co-localization. Scale bars: 20 µm.