Fig 1: Effects of SMR, Mortalin, and Vimentin peptides on tumor cell exosome secretion in MDA-MB-231 and MCF-7 breast cancer cells. MDA-MB-231 and MCF-7 BC cells (200 µL, 1 × 104 cells/mL) were seeded into a 96-well plate and treated with 100 ng/mL of either wild-type (wt) or mutant (mut) peptides of SMR, Mortalin, or Vimentin. Treatments were performed in RPMI-1640 medium without transfection, and cells were incubated at 37 °C for 24 h. The cells were then treated with 0.5 µM/mL of N-Rh-PE, incubated at 4 °C for 1 h, and washed with PBS. Fresh medium was also added. The cultures were incubated at 37 °C for an additional 24 h. N-Rh-PE refers to N-(Lissamine Rhodamine B sulfonyl) phosphatidyl-ethanolamine, a fluorescent lipid dye commonly used to label membranes and track vesicle trafficking. (A) MDA-MB-231 Cells: Data analysis of exosome release revealed significant differences: UT vs. SMRwt, Mort #61/62(B)-wt, VIM-wt **** p < 0.00005 in MDA-MB-231. (B) MCF-7 Cells: Data analysis of exosome release revealed significant differences: UT vs. SMRwt, Mort #61/62(B)-wt, VIM-wt **** p < 0.00005 in MCF-7. Exosome secretion was assessed by measuring the amount of N-Rh-PE, which corresponds to the number of exosomes released into the extracellular medium. Bar graphs show the relative exosome release level. Data represent the mean ± SD of three independent experiments.