Fig. 3.

CQ accumulates within enlarged MLB/MVB-positive vesicles. MDCK cells were treated with 10 μM CQ for 12 h, which primes them for vacuolar expansion and MLB/MVB formation, followed by 100 μM CQ for 2 h before imaging. For fluorescence microscopy, cells were incubated with 0.5 μM LTG for 30 min immediately before imaging. A, LTG fluorescence (top) and the corresponding brightfield image (middle) of a representative CQ-treated cell was merged (bottom), showing the highly heterogeneous LTG fluorescence associated with MLB/MVBs within the expanded cytoplasmic vesicles. Scale bar, 4 μm. B, analysis of intracellular CQ distribution by confocal Raman microscopy. Top, brightfield image showing 100 μM CQ-treated (a) and untreated (b) cells from which spectra were acquired. Scale bar, 5 μm. Bottom, spectrum 1 was acquired from 100 mM CQ solution in buffer, as reference. Spectra 2 and 3 were acquired from the vesicles and cytosol of treated cells, respectively; spectra 4 and 5 were acquired from the vesicles and cytosol of untreated cells. In these spectra, CQ-specific Raman vibrational peaks (around wavenumbers 1370 and 1560) were identified on the basis of spectrum 1. The CQ-specific Raman signal was mostly localized within the expanded vesicles of CQ-treated cells.