Figure 2. Effect of S. haenkei treatment on growth arrest and senescence in Pten−/− MEFs.

(a) Proliferation of Pten−/− MEFs in culture after 5 days of treatment with S. haenkei extract. Pten−/− MEFs were plated in concentration of 2×10⁴ cells/ml and treated for 5 days with 10μM MDM2i (Nutlin-3) or 10μg/ml SH extract. After this period, the proliferation was determined using Crystal violet staining. (c) Results are expressed as mean values (+SEM) of absorbance at 590nm for duplicates treated with SH and triplicate for control and Nutlin-3 treated groups, from one representative experiment out of 3 independent experiments. (b-d) Senescence of Pten−/− MEFs in culture after 5 days of treatment with S. haenkei extract. The graph represents percentage of β-galactosidase positive cells revealed in culture upon 5 day treatment with 10μM MDM2i (Nutlin-3) or 10μg/ml S. haenkei extract. Quantifications were done on 4 images (roughly 500 cells) per experiment by determining the ratio of perinuclear blue–positive to perinuclear blue–negative cells. Results are expressed as mean values (+SEM) of cell count in three independent experiments.