Figure 2. sEVs secreted from senescent cells promote the proliferation of MCF-7 cells.

(a) Relative numbers of MCF-7 cells grown in the presence of CM compared with the number of cells grown in normal medium (DMEM supplemented with 10% FBS). CM was prepared from control (early passage) and replicative senescent (late passage) TIG-3 cells, or control and DXR-induced senescent RPE-1 cells, and was used directly or after depleting EVs by ultracentrifugation. Cell densities of early and late passage TIG-3 cells were 8 × 10² cells cm⁻² at 1 day before starting CM preparation. Cell densities of control and DXR-induced senescent RPE-1 cells were 1.4 × 10⁴ cells cm⁻² at 1 day before starting CM preparation. MCF-7 cells were plated at a density of 4 × 10² cells cm⁻² 1 day before starting the experiments. The basal proliferation rate, that is, the fold increase in cell number of untreated MCF-7 cells at culture day 3, was 2.03. The CM to be used for cell culture was prepared by culturing the cells in normal medium for 3 days. Statistical analysis was applied only to the data of culture day 3. (b) Relative numbers of MCF-7 cells grown in the presence of sEVs prepared from control or DXR-induced senescent RPE-1 cells compared with the number of cells grown in normal medium. Pre-senescent RPE-1 cells were subconfluent and DXR-induced senescent RPE-1 cells were at a density of 1.4 × 10⁴ cells cm⁻² at 1 day before starting CM preparation. MCF-7 cells were plated at a density of 4 × 10² cells cm⁻² 1 day before starting the experiments. sEVs were added to the medium at a concentration of 2 × 10⁹ particles per ml. Statistical analysis was applied only to the data of culture day 3. Replicates are biological replicates (n=3). Error bars indicate s.d. *P<0.05 (one-way ANOVA with the post-hoc Dunnett's two-tailed test for a and two-tailed t-test for b).