Extended Data Fig. 7: Cytoplasmic delivery of MC1_Cy5.5 is not mediated by the fluorophore and is independent of endocytosis.

a, Free Cy5.5 dye accumulates in mitochondria. Upper panels: KPL-4 cells were pre-incubated with Mitotracker^TM Red FM (MTR, shown in the green channel) for 20 min, washed twice, then free, capped Cy5.5 dye (red channel) was added for 9 min and imaged. Lower panels: cells were pre-loaded overnight with Oregon Green dextran (Dex, lower panels), washed, then free Cy5.5 dye was added for 6 and imaged. The free Cy5.5 fluorophore colocalizes perfectly with the mitochondrial MitoTracker^TM, clearly distinct from the fluid phase endocytic marker, dextran, and is absent from the cytoplasm (in contrast to MC1_Cy5.5). Scale bar is 40 µm. b, Three independent endocytic inhibitors prevent endosomal uptake of MC1_Cy5.5 with no detriment to its cytoplasmic delivery. KPL-4 cells were preincubated for 6 min in serum-free media with the dynamin inhibitor Dynasore (which inhibits clathrin-mediated and caveolar endocytosis); or for 30 min with 2% methanol (vehicle), 5 µg ml⁻¹ cholesterol extractor filipin (an inhibitor of lipid raft and caveolar endocytosis), or 200 µg ml⁻¹ dimethyl amiloride (DMA), a micropinocytosis inhibitor. Cells were then incubated for a further 30 min with 2 µM MC1_Cy5.5 and fresh inhibitors before washing, fixing and imaging. Scale bar is 40 µm. Panels are representative of at least 2 experiments. All three inhibitors prevented endocytosis of MC1_Cy5.5, as evidenced by loss of punctate endosomal signal (confirming that uptake in the vehicle control is mediated by nonspecific fluid phase endocytosis), but none of them prevented MC1_Cy5.5 accumulation in the cytoplasm. Data are representative of 2 independent experiments.