[16]. Thus, we suspect that BE(2)-C neuroblastoma cells may provide a specific cellular environment (e.g., co-factors) necessary for the four HOXD proteins to exert their functions. Together, these observations make neuroblastoma cells an attractive system for uncovering the molecular mechanisms that govern the functions of HOXD proteins in regulation of neuroblastoma cell proliferation and neuronal differentiation. Materials and Methods Cell culture and inducible expression of HOXD genes The human neuroblastoma cell line BE(2)-C (CRL-2268, ATCC) was cultured in a 1:1 mixture of DMEM and Ham’s nutrient mixture F12 supplemented with 10% fetal bovine serum (Invitrogen). The Retro-X Tet-Off Advanced Inducible Gene Expression System (Clontech) was used to generate BE(2)-C- derived cell lines with inducible expression of individual HOXD genes in the absence of doxycycline. Myc-tagged human HOXD genes were generated by PCR, verified by sequencing, and subcloned into pRetroX-Tight-pur. cDNAs coding for individual HOXD proteins were obtained from Open Biosystems, OriGene, ATCC, Addgene, and Invitrogen. Retroviruses were produced in 293FT cells using the packaging plasmids pHDM-G and pMD.MLVogp. For morphologic examination of neuronal differentiation, cells were examined and phase contrast images captured daily using an Axio Observer microscope and the software AxioVision (Carl Zeiss MicroImaging). RA treatment All trans-RA (Sigma-Aldrich) was dissolved in DMSO and 10 mM stock solutions were prepared. BE(2)-C cells were treated with 10 mM RA. DMSO at the final concentration of 0.1%was used as negative control (untreated). Cells were examined and phase contrast images captured using an Axio Observer micro- scope and the software AxioVision. Fluorescence-activated cell sorting (FACS) Cells were fixed in 70%ethanol, incubated with ribonuclease A (Sigma-Aldrich), and stained with 20 mg/ml propidium iodide (Invitogen). Samples were analyzed with a FACSCalibur system and ModFitLT V3.2.1 software (BD Bioscience). Quantitative reverse-transcription PCR (qRT-PCR) Total RNA was purified from cells using Trizol (Invitrogen). Reverse transcriptions were performed using SuperScript II Reverse Transcriptase (Invitrogen). Quantitative real-time PCR were performed using a RT2 SYBR green/Fluorescein PCR master mix (SABiosciences). The primer sequences for HOXD genes have been described previously [48] and for CCNB1, CDK1, HOXC9, NEFM, RET, and GAPDH are listed in Table S1. All primer pairs were verified by melting curve analysis following qRT-PCR, with each primer pair showing a single desired amplification peak. Samples from at least two independent experiments were analyzed by qRT-PCR and each sample was assayed in triplicate. PCR was performed on an iQ5 real-time PCR system (Bio-Rad) and the data from two or more independent samples were combined and analyzed using the GFX Manager Gene Expression Analysis software (Bio-RAD). Immunoblotting Cells were suspended in standard SDS sample buffer and protein concentrations were determined using a protein assay kit (Bio-Rad) with bovine serum albumin as reference. Proteins (50 mg) were separated on SDS-polyacrylamide gels, transferred to HOXD Regulation of Neuroblastoma Differentiation nitrocellulose membranes, and probed with the following primary antibodies: mouse anti-Myc-tag (9E10, hybridoma supernatant, 1:10), rabbit anti-cyclin A2 (sc-751, 1:200), mouse anti-cyclin B1 (sc-245, 1:200), mouse anti-cyclin D1 (sc-20044, 1:200), mouse anti-CDK1 (610037, 1:1000; BD Biosciences), rabbit anti-CDK4 (sc-260, 1:200), rabbit anti-CDK6 (sc-177, 1:200), rabbit anti- PHOX2B (1:1000) [49], mouse anti-NEFM (NF-09, sc-51683, 1:200), mouse anti-alpha-tubulin (B-5-1-2, 1:5000; Sigma-Aldrich), and rabbit anti-beta-actin (600-401-886, 1:2000; Rockland Im- munochemicals). Unless indicated, all primary antibodies were purchased from Santa Cruz Biotechnology. For detecting NEFM, PHOX2B, cyclin A2, cyclin D1, CDK4, CDK6, Myc-tag, alpha- tubulin and beta-actin, membranes were incubated with IRDye- labeled secondary antibodies and scanned on an Odyssey Infrared Imaging System (LI-COR Biosciences). For detecting cyclin B1 and CDK1, membranes were incubated with Horseradish peroxidase conjugated goat anti-mouse IgG (1:5000, Santa Cruz Biotechnology). Proteins were visualized using a SuperSignal West Pico chemiluminescence kit (Pierce). Immunofluorescence Cells were fixed with 4% paraformaldehyde and stained with rabbit anti-KI67 (sc-15402, 1:100) and mouse anti-NEFM (NF-09, sc-51683, 1:100). All secondary fluorescence antibodies were from Molecular Probes and used at 1:800 dilutions for goat anti-mouse (Alexa Fluor 488) and goat anti-rabbit (Alexa Fluor594). Nuclei were stained with DAPI. Fluorescent images were captured with a fluorescence microscope (Carl Zeiss Axio Observer). RNA interference Retroviral (pSM2) constructs expressing shRNA to human HOXD8 (RHS1764-9501133) or HOXD9 (RHS1764-9691903) were purchased from Open Biosystems and the shRNA-coding sequences were subcloned into the retroviral shRNAmir Tet-Off vector [50] for inducible expression in BE(2C)-C/Tet-Off cells in the absence of doxycycline [25]. The luciferase shRNAmir Tet- Off construct was used as control. Retroviruses were produced in 293FT cells using the packaging plasmids pHDM-G and pMD.MLVogp. Chromatin Immunoprecipitation (ChIP) ChIP assays were performed on BE(2)-C/Tet-Off/myc- HOXD8 cells cultured in the presence (2 mg/ml) or absence of doxycycline for 3 days, using mouse anti-Myc tag (clone 4A6, Millipore), rabbit anti-H3K4me3 (07–473, Millipore), or control mouse or rabbit IgG according to the online protocol (http://jura. wi.mit.edu/young_public/hESregulation/ChIP.html). Two inde- pendent ChIP genomic DNA samples were analyzed and each sample was assayed in triplicate by qPCR. Primer sequences for ChIP-qPCR assays are listed in Table S2. Quantification and statistical analysis For KI67 immunofluorescence staining, approximately 400 cells (DAPI-positive) were counted from at least 5 randomly selected 400x fields independently by three investigators, and the percentage of KI67+ cells was determined. All quantitative data including cell cycle analysis, KI67 staining, and qRT-PCR were analyzed between test and control groups using two-tailed Student’s t-test, and p value ,0.05 was considered to be statistically significant. PLoS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e40728