Figure 3. Effect of S. haenkei treatment on replicative senescence in human fibroblasts.

(a) Growth curve of human WI38 fibroblasts treated with S. haenkei extract. WI-CCL75 human fibroblasts were plated 300.000 cells per 10cm dish, and subsequently passed and replated in the same number every 3 days for total of 24 passages up to the point when treatment with S. haenkei was initiated. At passage 25, cells were plated at the same number 300.000 cells per plate, and treated with 10μg/ml SH extract. Every 3 days cell number was determined by Trypan blue staining and cells replated 300.000 per plate and re-treated. Results are expressed as fold change in cell number from one representative experiment out of 4 independent experiments. (b) Senescence of human WI38 fibroblasts treated with S. haenkei extract. The graph represents percentage of β-galactosidase positive cells revealed in culture at each passage. 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 four independent experiments. (c) Cell death in culture of human WI38 fibroblasts upon treatment with S. haenkei extract. The graph represents percentage of Trypan blue positive (dead) cells revealed in culture at each passage. Quantifications were done on one experimental image (roughly 100 cells) in one representative experiment.