(A–C) Number of SKP spheres generated from secondary human SKPs grown for 7 days in varying concentrations of alprostadil (Alp) (A) or TM (B) or in 100 nM alprostadil, TM, kaempferol (Kae), MG-624, or pramoxine (Pram) (C). In (C) numbers are expressed relative to DMSO alone.

(D–F) Number of SKP spheres generated from secondary neonatal rat SKPs grown for 7 days in 100 nM of each of the five drugs (D), or in varying concentrations of alprostadil (E) or TM (F).

(G and H) Number (G) and size (H) of rat SKP spheres generated over 7 days in 100 nM alprostadil, TM, or both.

(I) Number of rat SKP spheres generated in 14-day clonal methylcellulose assays with 100 nM alprostadil, TM, or latanoprost (Latan).

(J and K) Secondary rat SKP spheres were grown for 4 days, and 100 nM alprostadil or TM was added for two additional days. (J) shows spheres immunostained for Ki67 (top) and counterstained with DAPI to show nuclei (bottom). (K) shows the percentage of Ki67-positive cells. Scale bar represents 100 μm.

(L) NIH 3T3 cells were treated for 24 hr with 100 nM alprostadil or TM, immunostained for Ki67, and the percentage of positive cells determined. N.S., not significant.

In all panels, results were pooled from 3 to 4 independent experiments with, in the human experiments, 3–4 different human SKP lines.