9-cis retinoic acid (RA) suppresses OTX2 expression better than other available retinoids. (A) Expression of OTX2 in MB cell lines evaluated by RT-PCR. OTX2 or GAPDH primers were used with cDNA from the 11 MB cell lines for 28 cycles of PCR. (B) 9-cis RA suppresses OTX2 expression better than other retinoic acids. A panel of RAs at 0.1 µM was incubated with D425 cells for 48 hours. Protein level of OTX2 was analyzed by Western blotting. Equal loading was confirmed by probing with an anti-GAPDH antibody. Other retinoids tested were: ATRA, all trans-RA; 13-cis, 13-cis RA; AC, acitretin; MA, methoprene acid; TTNPB, 4-[E-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid. (C) Comparison of ATRA, 9-cis RA, and AM580 in suppressing OTX2 expression. D425 cells were incubated with 0.1 or 0.5 µM of the indicated retinoids for 48 hours. OTX2 protein level was detected by Western blotting. (D) Comparison of RAs in inhibiting the growth of D425 cells. A volume of 0.1 µM of the indicated RAs was incubated with D425 cells for 6 days with a re-addition of RAs after 3 days. Viable cells were monitored by cell proliferation assay reagent WST-1 and calculated in percentage to the DMSO control. RAHA, retinoic acid p-hydroxyanilide; GA, geranylgeranoic acid.