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. 2010 Feb 5;12(7):655–663. doi: 10.1093/neuonc/nop062

Fig. 2.

Fig. 2.

Suppression of MB cells by RA is found only in cells expressing OTX2, and RA sensitivity is lost with exogenous OTX2 expression. (A) Comparison of the growth inhibition from ATRA and 9-cis RA on MB cell lines. Medulloblastoma cell lines (D283, D341, D425, DAOY, UW228, and Mhh1) were incubated with DMSO (control), 0.01, 0.1, 1, or 10 µM of the indicated RA for 6 days. After 3 days, we re-added RA. Viable cells were monitored by WST-1 and calculated in percentage to the DMSO control. All the OTX2-positive cells (D283, D341, and D425) were inhibited by ATRA and 9-cis RA, while 9-cis RA generally exhibited inhibition at slightly lower concentration in D425 cells. (B) Overexpression of exogenous OTX2 reduces the growth inhibitory effect of 9-cis RA on D425 cells. OTX2 was subcloned in fusion with 3 copies of FLAG tag (3F-OTX2) and stably transfected in D425 cells. Single clones were selected by limited dilution and tested for 3F-OTX2 expression by Western blotting. D425 cells with vector alone were used as a negative control. On the anti-OTX2 Western blot, 3F-OTX2 showed a slower migration pattern compared with that of the endogenous OTX2 protein. Clone 5 (Cl. 5), Clone 20 (Cl. 20), and control cells were incubated with DMSO, 0.01, 0.1, 1, or 10 mM 9-cis RA for 96 hours. Viable cells were measured by WST-1 and graphed in percentage to the DMSO control (0 hour).