RNA extraction, RT-PCR and real-time PCR. Total RNA were extracted using

Transcription

1 Supplementary Information Materials and Methods RNA extraction, RT-PCR and real-time PCR. Total RNA were extracted using Trizol reagent (Invitrogen,Carlsbad, CA) according to the manufacturer's instructions. 2 µg of total RNA was used for cdna synthesis with random hexamers. For RT-PCR amplification of HOXB7, an initial amplification using HOXB7 primers was done with a denaturation step at 95 C for 10 min, followed by 28 cycles of denaturation at 95 C for 1 min, primer annealing at 55 C for 30 s, and primer extension at 72 C for 45s. Upon completion of the cycling steps, a final extension at 72 C for 5 min was done and then the reaction was stored at 4 C. Real-time PCR was carried out using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Reactions were run in triplicate in three independent experiments. The geometric mean of housekeeping gene GAPDH was used as an internal control to normalize the variability in expression levels. The primer sequences are provided in Supplemental table 1. Expression data were normalized to the geometric mean of housekeeping gene GAPDH to control the variability in expression levels and were analyzed using the 2 -ΔΔCT method described by Livak and Schmittgen (1). Immunohistochemistry (IHC). IHC staining and scoring were done as previously described (2). Tissue sections were incubated with polyclonal mouse antibody against HOXB7 (Sigma-Aldrich, MO) at dilutions of 1:200 overnight at 4. For negative controls, the mouse anti-hoxb7 antibody was replaced with normal nonimmune

2 serum. The cells at each intensity of staining were recorded on a scale of 0 (no staining), 1 (weak staining = light yellow), 2 (moderate staining = yellowish brown), and 3 (strong staining = brown). An intensity score of 2 with at least 50% of malignant cells with positive HOXB7 staining was used to classify tumors with high expression, and < 50% of malignant cells with nuclear staining or < 2 intensity score classified tumors with low expression of HOXB7. Mouse anti-ki-67 monoclonal antibody (Dako, Copenhagen, Denmark) was used to evaluate the Ki-67 labeling index, which was calculated as the positive tumor nuclei divided by the total number tumor cells in each case. The cut-off value was based on a measurement of heterogeneity with a log-rank test statistical analysis with respect to overall survival. Using this assessment system, 30% was identified as an optimal cutoff value for Ki-67 labeling index. 3-(4,5 -Dimethyl-2-thiazolyl)-2,5 -diphenyl-2h-tetrazolium bromide (MTT) assays. Cells were seeded on 96-well plates at initial density of ( /well). At each time point, The cells were stained with 100μl sterile MTT dye (0.5 mg/ml, Sigma-Aldrich, MO) at each time point for 4 h at 37ºC followed by removal of the culture medium and addition of 150 μl of dimethyl sulphoxide (DMSO) (Sigma-Aldrich, MO). The absorbance was measured at 570 nm, with 655 nm as the reference wavelength. All experiments were performed in triplicates. The data were analyzed by factor analysis and one-way ANOVA. Cell cycle analysis. Cell cycle distribution was examined by measuring the cellular DNA content using flow cytometry. Cells at 80-90% confluence were incubated for

3 36 h in the RPMI-1640 medium containing 0.5% FBS, then released through culturing back in RPMI-1640 medium with 10% FBS for 12 h. 1x10 6 cells were collected and fixed with 70% cold ethanol. After treated with RNase A (10 μg/ml) for 30 minutes at 37 C, the cells were resuspended in 0.5 ml propidium iodide (PI) solution (50 μg/ml in 0.1% sodium citrate with 0.1% NP-40). Cell cycle distribution was analyzed by FACScan cytometry (Becton-Dickinson, San Jose, CA, USA). References: 1. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25: Liao WT, Wang X, Xu LH, et al. Centromere protein H is a novel prognostic marker for human nonsmall cell lung cancer progression and overall patient survival. Cancer 2009;115:

4 Supplementary Table Supplementary Table S1. Primer Sequences Used for Reverse Transcription-Polymerase Chain Reaction (PCR) and Real-time Quantitative Reverse Transcription-PCR (5' to 3') Gene Forward primer Reverse primer Probe RT-PCR HOXB7 AGAGTAACTTCCGGATCTA TCGGCTTCAGCCCTGTCTT GAPDH CCACCCATGGCAAATTCCATGGCA TCTAGACGGCAGGTCAGGTCCAC HOXB7 AGAGTAACTTCCGGATCTA CAGGTAGCGATTGTAGTG FAM-ACCCCTGGATGCGAAGCTCA-TAMRA Real-time PCR p27kip1 CCGGTGGACCACGAAGAGT GCTCGCCTCTTCCATGTCTC FAM-AACCCGGGACTTGGAGAAGCACTGC-TAMRA cyclind1 CCGTCCATGCGGAAGATC ATGGCCAGCGGGAAGAC FAM-CTTCTGTTCCTCGCAGACCTCCAGCAT-TAMRA GAPDH GACTCATGACCACAGTCCATGC AGAGGCAGGGATGATGTTCTG FAM-CATCACTGCCACCCAGAAGACTGTG-TAMRA

5 Supplementary Table S2. Correlation between Clinicopathologic Features and HOXB7 expression in CRC Characteristics HOXB7 Expression Low High Age mean(56) > mean (56) Gender Male Female Histology Columnar adenocarcinoma Mucinous adenocarcinoma Others 11 7 Pathologic stage ~ Dukes Stage A 14 2 B C D T stage 1~ N stage Distant metastasis 0 (no) (yes) Ki-67 labeling index <30% % P <

6 Supplementary Table S3. Univariate and Multivariate Analyses of HOXB7 expression level in different prognostic parameters in patients with CRC using Cox Regression model Variable T stage Pathologic stage HOXB7 Category No. Patients 1~ ~4 44 Low 103 High 121 Univariate analysis P Regression Coefficient (S.E) P Multivariate analysis Relative Risk 95% Confidence Interval < (0.183) < < (0.177) < (0.225)