Extended Data Fig. 2. Intracellular fusion of R18-labeled liposomes is pH-dependent.

a, Liposome fusion (red) in Huh7 cells treated with 2 μM Monensin, 30 μM Chloroquine diphosphate, or 5mM Amonium chloride (NH₄Cl) prior to the addition of PS:PC:Chl-R18 liposomes. Nuclei were stained with DAPI (blue). Cells were analyzed in a LSM 700 confocal microscope. Micrographs were taken using a 40× oil objective. Scale bars represent 50μm. b, Quantitative analysis of the fusion of R18-labeled liposomes in endosomes from (a). The R18 fluorescence intensity (red) of 20 cells from (a) was measured using ImagJ software and the experiment was repeated 3 times (n=60). Box and whiskers plot was done using the Tukey method. Box limits, upper and lower quartiles; Center line, median; whiskers, 1.5× interquartile range; points, outliers; P values between untreated and treated cells were determined by two-sided Mann-Whitney test. c, Lack of cytotoxicity of the compounds was confirmed by flow cytometry using a caspase-3 apoptosis assay based on the irreversible binding of cell-permeable inhibitor DEVD-fmk conjugated to sulfo-rhodamine (Red-DEVD-fmk) to activated caspase-3. Stained cells were analyzed with a FACSCanto II (BD Biosciences) using FlowJo v8.5 software. Apoptotic/dead Huh7 control cells were prepared by heat treatment at 55ºC for 7 min. d, Liposome fusion with intracellular membranes. Huh7 cells were treated with PS:PC:Chl-R18 liposomes (R18 liposome fusion events, red) for 15 min at 37ºC, the plasma membrane was stained with CellMask (green) for 15 min at 37ºC, and nuclei were stained with DRAQ5 (blue). Cells were analyzed in a LSM 700 confocal microscope. Micrographs were taken with 40× oil objective. Scale Bars represent 25μm. Liposome fusion events detected by R18 fluorescence (red) that did not co-localize with plasma membrane stained with Cell Mask (green). Results are representative of 3 independent experiments.