Fig. 3.

Effects of PI3K and Akt inhibitors on human foreskin-derived skin-derived precursors (hSKPs). (A) Western blot results showing Akt phosphorylation in hSKPs cultured with 0.1% DMSO as a control and 10 μm LY294002 (the left panel) or 5 μm Akt inhibitor VIII (the right panel). Histograms show statistical results of gray-scale analysis of three independent cultures and representative immunoblots are attached below. Asterisk means P < 0.05 when compared with control. (B) MTS assays of hSKPs treated with DMSO (as a control), LY294002 or Akt inhibitor VIII for 48 h. The statistical results were derived from three independent experiments. Asterisk means P < 0.05 when compared with control. (C) Proliferation of hSKPs after treatment with DMSO (as a control), LY294002 or Akt inhibitor VIII for 48 h shown by Ki67 staining. Left panels show representative hSKP sphere staining and the histogram on the right shows the statistical results from three independent cultures. Asterisk means P < 0.05 when compared with control. (D) SA-β-Gal staining of hSKPs cultured with DMSO (as a control), LY294002 or Akt inhibitor VIII for 12 days. Upper panels show representative staining in different groups and lower histograms show the percentages of three types of spheres described previously. (E) Western blot assay showing relative expression of p53 (the left panel) and p16 (the right panel) in hSKPs cultured with DMSO (as a control), LY294002, or Akt inhibitor VIII for 12 days. Histograms show the results of gray-scale analysis and representative immunoblots are attached below. (F) Morphology of hSKP spheres formed in conditions with DMSO (as a control), LY294002 or Akt inhibitor VIII in a typical culture. Group names are labeled on the top and mean diameters of spheres are given at the bottom. (G) Sphere-forming efficiency of hSKPs cultured with DMSO (as a control), LY294002 or Akt inhibitor VIII in liquid culture system. The efficiency was presented as number of spheres formed form 10⁴ cells. (H) Sphere-forming assay of hSKPs treated with inhibitors of the PI3K-Akt pathway in semisolid media containing methylcellulose. (a) Number of spheres with diameters from 30 to 50 μm formed from 10⁴ hSKPs in the presence of 0.1% DMSO (control), 10 μm LY294002 or 5 μm Akt inhibitor VIII. (b) Number of spheres with diameters > 50 μm formed from 10⁴ hSKPs in the presence of 0.1% DMSO (control), 10 μm LY294002, or 5 μm Akt inhibitor VIII. (c) Number of spheres with diameters > 30 μm formed from 10⁴ hSKPs in the presence of 0.1% DMSO (control), 10 μm LY294002 or 5 μm Akt inhibitor VIII. Sphere number of each treatment in c equals the combination of those in a and b. Data show the results derived from a 25-year-old donor (blue columns) and a 31-year-old donor (yellow columns). Each treatment had triplicates, and results were statistically analyzed. Asterisk means P < 0.05 when compared with control. MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).