Figure 8.

Bacoside A induced enlarged vacuoles are macropinosomes that undergo time dependent pre-lysosomal fusion. (A) Confocal images of Bacoside A (12 h) treated LN229 glioblastoma cells showed large vacuoles positive for dextran-TMR (red), a marker of macropinosomes. Insets in the panel shows that in 24 h Bacoside A treated LN229, cells had rounded off and were filled with dextran-TMR, delivered by macropinosomes to the cytoplasm. Hence, cell swelling was a net consequence of excess fluid uptake by the tumor cells. The white arrowhead points to a necrotic cell attached to swelled cell with squashed nucleus (cyan/white). The vacuoles were identified to be positive for late endosome/macropinosome markers. (B) Rab7 and (C) LAMP1. White arrows in panels (B,C), points to the localization of Rab7 and LAMP1 surface proteins to the membranes of vacuoles. (D) shows Dextran-TMR positive macropinosome surface positivity for Rab7 (green). Similarly, (E) shows Dextran-TMR positive macropinosome surface positivity for LAMP1 (green) but (F) not for LC3II, an autophagosome marker (also see Supplementary Figure S8). (G) The Bacoside A treated LN229 tumor cell vacuolar membranes were also positive for caveolin-1 protein and in many vesicles it was noticed in the vesicle lumen, suggestive of both caveolar/surface origin of vacuoles and non-caveolar surface origins from the caveolar flattened areas respectively. Overall, treated cells showed major loss of surface CAV1 in comparison to untreated LN229 cells. The images are representative of five independent experiments.