HOXD Regulation of Neuroblastoma Differentiation Figure 4. Distinct functions of HOXD proteins in regulation of neuronal differentiation. (A–B) Phase contrast imaging (A) and NEFM immunofluorescent staining (B) of BE(2)-C/Tet-Off/myc-HOXD cells cultured in the presence or absence of Doxy for 7 days. Scale bars, 50 mm. Shown are representative of three independent experiments with similar results. (C) Immunoblot analysis of PHOX2B and NEFM protein levels in the same cell samples described in Figure 3. Beta-actin levels are shown as loading control. Shown are representative of two independent experiments with similar results. (D) Quantification of NEFM mRNA levels in BE(2)-C/Tet-Off/myc-HOXD cells cultured in the presence or absence of Doxy for 3 days. The NEFM mRNA levels in BE(2)-C/Tet-Off/myc-HOXD cells in the presence of Doxy were designated as 1.0 (dashed line). The data were from two independent samples with each being assayed in triplicates and analyzed using two-tailed Student’s t-test with the p values indicated. Error bars, SD. doi:10.1371/journal.pone.0040728.g004 PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e40728