Fig 4. Activating endocytosed INF7 with acetic acid causes endosomal escape of SR into the cytosol.

A. Liposomes containing SR with or without INF7 were incubated with CV1 cells for 1 hr, washed with Ca²⁺/Mg²⁺-free HBSS containing 1 mM EDTA, and imaged by fluorescence microscopy (Pre Acid); endosomal SR appears as fluorescent puncta. After the addition of 1% acetic acid to the extracellular solution, fluorescence in cells incubated with INF7-containing liposomes (+INF7) becomes cytosolic and more homogeneous. In contrast, in cells incubated with liposomes lacking INF7 (-INF7), the appearance of SR fluorescence is unchanged by acidification. B. Time course showing changes in SD of pixel intensity relative to mean intensity (σ/F, normalized to mean baseline value before acidification at time 0). Time of addition of acetic acid is indicated by the black arrowhead. Activated INF7 rapidly releases SR into the cytosol, and the more homogeneous distribution of SR leads to lower SD values than in the case of incubation without INF7, where SR remains confined to endosomal compartments. Also see S1 and S2 Videos in Supporting Information.