Structures labeled by mepacrine but not by LysoTracker are first observed in proplatelets. (A-B) G1ME2 cells differentiated for 5-6 days to proplatelets were incubated with 50 μM mepacrine (green) and 200 nM LysoTracker Red DND-99 (red) for 30 min. The panels show raw images of 2 G1ME2-derived proplatelet strings (G1ME2-proplatelet), magnified from the boxed region shown in the corresponding DIC images; the dotted white line in the fluorescent images indicates the outline of the proplatelet from the DIC image. Panel A depicts a proplatelet string with distinct mepacrine-containing compartments (arrows, in insets), and panel B demonstrates a proplatelet string only with puncta that label with both mepacrine and LysoTracker Red (arrowheads, in insets). Scale bars, 5 μm. (C) The number (mean ± SD) of total puncta and puncta labeled by mepacrine alone (mep-only puncta) or by both mepacrine and LysoTracker (coloc. puncta) were quantified per proplatelet string identified as detached or attached as indicated in the text. (D) Quantification of the percentage (mean ± SD) of mepacrine (Mep) labeling colocalized with LysoTracker Red (LyTrR) in detached (n = 42) and attached (n = 30) proplatelets, normalized to 100% for overlap of LysoTracker Red with LysoTracker Green DND-26 (LyTrG) in G1ME2-MK (n = 79). Proplatelets were imaged by spinning-disk confocal microscopy at room temperature with a Leica DMi8 inverted microscope equipped with a Hamamatsu Orca Flash 4 CMOS camera, a 100× Plan Apochromat objective lens (1.4 NA), and VisiVIEW imaging software. Imaging medium was IMDM. Mepacrine has an excitation/emission of 436/525 nm; LysoTracker Red excitation/emission: 577/590 nm. **P < .01. int., intensity.