HOXD Regulation of Neuroblastoma Differentiation Figure 5. HOXD8 is a mediator of RA action. (A) qRT-PCR analysis of HOXD8 mRNA levels in BE(2)-C/Tet-Off cells with inducible expression of an shRNA sequence against Firefly luciferase (Luc-sh) or human HOXD8 (HOXD8sh-1) in the absence of Doxy for 6 days. The HOXD8 mRNA levels in BE(2)- C/Tet-Off/luc-sh cells in the presence of Doxy were designated as 1.0. The data were from two independent samples with each being assayed in triplicate and analyzed using two-tailed Student’s t-test with the p values indicated. (B–D) Phase contrast imaging (B) and cell cycle analysis (C–D) of BE(2)-C/Tet-Off/Luc-sh and BE(2)-C/Tet-Off/HOXD8sh-1 cells that were cultured in the absence of Doxy for 6 days and then either untreated or treated with 10 mM RA for 3 or 6 days. The data in (D) were from three independent experiments and analyzed using two-tailed Student’s t-test with the p values indicated. (E) qRT-PCR analysis of the mRNA levels of CCNB1, CDK1, and RET in BE(2)-C/Tet-Off/Luc-sh and BE(2)-C/Tet-Off/HOXD8sh-1 cells that were cultured in the absence of Doxy for 6 days and then treated with 10 mM RA for 6 days. The mRNA levels of the same genes in BE(2)-C/Tet-Off/ Luc-sh cells cultured in the absence of Doxy without RA treatment were designated as 1.0 (dashed line). The data were from two independent samples with each being assayed in triplicate and analyzed using two-tailed Student’s t-test with the p values indicated. Error bars (A, D, and E), SD. doi:10.1371/journal.pone.0040728.g005 according to their location within the four (HOXA-D) clusters, in a time- and RA concentration-dependent fashion. This order corresponds to the expression patterns in developing axial systems in the human and mouse where 39 genes are expressed earlier and 59 genes later [28,29]. These findings indicate that the temporal response of HOX genes to RA in human embryonal carcinoma cells is of physiological relevance, leading to the suggestion that a molecular understanding of the RA response in cell-based systems may provide important insights into the temporal regulation of HOX gene expression in the embryo [41]. In NT2/D1 cells, RA induces all HOXB genes as well as 39 HOXA, HOXC and HOXD genes roughly corresponding to the HOX-5 paralog groups [11,12]. This is in striking contrast to BE(2)-C cells in which RA induces all HOXD genes, which largely recapitulates the sequential activation of Hoxd genes observed in the mouse embryo [42]. Thus, RA induction of HOXD genes in neuroblas- toma cells represents a valuable model for studying the molecular mechanism that controls the temporal expression of HOXD genes. Studies with Drosophila Hox genes have provided convincing evidence that each Hox gene specifies a unique morphology along the anteroposterior axis. Therefore, replacing one Hox gene with another will switch the identity of the tissue in which it is expressed PLoS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e40728