HOXD Regulation of Neuroblastoma Differentiation Figure 2. Distinct functions of HOXD proteins in regulation of cell proliferation. (A–B) Immunofluorescent staining (A) and quantification (B) of KI67-expressing BE(2)-C/Tet-Off/myc-HOXD cells cultured in the presence (Doxy+, 2 mg/ml) or absence doxycycline (Doxy-) for 6 days. Scale bars, 50 mm. The data in (B) were obtained from at least 5 randomly selected 400x fields (,400 DAPI-positive cells) and analyzed using two-tailed Student’s t-test with p values indicated. Error bars, SD. (C) FACS analysis of the cell cycle status of BE(2)-C/Tet-Off/myc-HOXD cells cultured in the presence or absence of Doxy for 6 days. Shown are representative of two independent experiments with similar results. doi:10.1371/journal.pone.0040728.g002 a BE(2)-C-derived cell line with inducible expression of an shRNA sequence against the human HOXD9 cDNA, which displayed a marked reduction (69%) in the expression of HOXD9 mRNA in the absence of doxycycline (Figure S4 A). HOXD9 knockdown had no significant effect on the ability of RA to induce morphologic differentiation and G1 arrest (Figure S4 B–D), demonstrating that HOXD9 is not required for RA action, at least in BE(2)-C neuroblastoma cells. These findings also provided PLoS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e40728