Fig. 6.

Induction by WT1-pTj of cellular senescence in human melanoma cells. (A) Quantification of colonies formed after removal of the peptides and TMZ. A2058 cells were treated with WT1-pTj, C PEP and TMZ for 4 days. Cells were detached and equal numbers of untreated and treated cells were seeded and cultivated in fresh media for additional 4 days. The number of colonies was scored in three independent experiments performed in duplicates; ^*p < 0.01 compared to Control and C PEP. (B) Representative photomicrographs (original magnification 400×) of A2058 cells treated with 0.5 mM WT1-pTj, C PEP or TMZ (positive control) for 4 days. Thereafter the peptides and TMZ were washed out and cells were stained for SA-β-Gal after incubation for additional 3 days in fresh media. Positive cells for SA-β-Gal were visualized under a phase contrast microscope and expressed as percentage of total cells counted at random. (C) Acridine orange was used to stain acidic vesicular organelles in untreated control cells (a); serum starved cells for 24 h (positive control) (b); cells treated with 0.5 mM WT1-pTj (c); cells treated with C PEP (d), for 4 days. Images are representative of two independent experiments performed in duplicates. Bars = 10 μM.