Retinoic acid inhibits proliferation and induces apoptosis. (a) AtT-20 cells were treated for 3 days with retinoic acid (RA). Proliferation was measured by ³H-thymidine incorporation. (b) AtT-20 cells were treated for 24 hours with 10 nM retinoic acid and seeded in soft agar. After 20 days, the colonies were stained with MTT and photographed. The control shows colonies composed of more than 300 cells. Retinoic acid–treated cells formed colonies composed of less than ten viable cells. (c–h) AtT-20 cells were stained with annexin V-FITC and photographed under ultraviolet (c, e, and g) or white light (d, f, and h). (c and d) Cells were treated with 1 μM H₂O₂ as a positive control for annexin staining induced by apoptosis. Cells were treated with 10 nM retinoic acid for 6 hours (e and f) or 24 hours (g and h). (i) AtT-20 cells were treated with 10 nM retinoic acid. At different times cells were collected, and their vitality was assessed by acridine orange–ethidium bromide staining. Caspase-3 activity was measured as indicated in Methods. (j) Cell viability was measured after 7 days using the WST-1 assay. The dotted line indicates the WST-1 values at the beginning of the treatment. Averages of four wells per treatment and SEs from one representative experiment are shown. *P < 0.01, **P < 0.001 compared with the corresponding basal or control values, determined by ANOVA in combination with the Scheffé test. The results are representative of four independent experiments.