ARPE-19 cells in culture were pretreated with 1 µM of U0126, for 1 h followed by incubation with 4HPR for additional 72 h. Total RNA extracted from treated cells was analyzed by real-time quantitative PCR as described under Materials and Methods. The values are mean ± SD, n = 4. *P < 0.001 compared with control; **P < 0.001 compared with 4HPR treatment. Panel a, Phase-contrast microscopic analysis of the inhibition of 4HPR-induced neuronal differentiation of ARPE-19 cells by U0126. Scale bar = 100 µm. Panel b, the inhibition of 4HPR-induced calretinin mRNA expression by U0126. Panel c, the inhibition of 4HPR-induced decrease of IGFBP5 mRNA expression by U0126. Panel d, the inhibition of 4HPR-induced decrease of IGFBP5 expression (protein) by U0126. Equal amount of serum-free culture medium from control and 4HPR treated ARPE-19 cells collected after 72 h was concentrated 50-fold, and then analyzed by Western blot using antibody specific for IGFBP5.