KIAA0101 mRNA and protein expressions were decreased with siRNA knockdown in the NCI-H295R adrenocortical carcinoma cell line.

<p><b>A</b>) H295R cells were transfected with KIAA0101 siRNA and negative control and analyzed for KIAA0101 mRNA expression after 48 hours of treatment. Columns represent KIAA0101 mRNA expression percentage relative to negative control ± standard deviation of four experiments. <b>B</b>) Representative Western blot demonstrating protein expression of KIAA0101 after 6 days of siRNA and negative control. <b>C</b>) Relative intensity of western blot using Image J Software. Columns indicate ratio of KIAA0101 and GAPDH intensity measurements. NC40 = Negative control at 40 nM; SI40  =  siRNA at 40 nM; NC80  =  Negative control at 80 nM; SI80  =  siRNA at 80 nM.</p

KIAA0101 siRNA knockdown reduced soft agar anchorage independent growth and invasion in NCI-H295R cells.

<p><b>A</b>) Cells cultured for 15 days in the presence of indicated concentrations of siRNA and negative control. Representative images are shown at 12X and 40X magnifications. <b>B</b>) The distribution and number of colonies in group treated with KIAA0101 siRNA and negative control group at 80 nM (p = 0.001; relative to negative control; NC80 =  Negative control at 80 nM; SI80  = siRNA at 80 nM). Error bars represent ± standard error of mean and is representative of four experiments. <b>C</b>) Invading cells were stained with 0.2% crystal violet and visualized by microscopy. Representative images at 20X following invasion assay. <b>D</b>) The distribution of number of cells in KIAA0101 siRNA and negative control group. Columns represent the mean ± standard deviation (SD) of three independent experiments performed in triplicate. *p<0.05 KIAA0101 siRNA vs. negative control. H295R cells were transfected with negative control or KIAA0101 siRNA at 80 nM for 120 h, followed by an invasion assay for 48 h.</p

Summary of sequencing changes in KIAA0101.

<p>*Percentage of mutation is for the samples which were analyzed by category and available for sequencing.</p><p>**ND (not detected).</p

Expression of KIAA0101 mRNA in ACC.

<p><b>A</b>) KIAA0101 mRNA expression in normal adrenal cortex (n  =  21), benign adrenocortical tumors (N  =  80), ACC (N  =  10), metastatic and recurrent ACC (n = 30) and the NCI-H295R cell line. KIAA0101 mRNA expression level was determined by quantitative RT-PCR and normalized to GAPDH mRNA expression. Columns represent mean ± standard deviation of replicate determinations. Statistical significant difference is indicated by an asterisk (*) (p<0.05, Kruskal Wallis test). Primary ACC vs. normal (p<0.0001), primary ACC vs. benign adrenocortical tumors (p<0.0001) and primary ACC vs. metastases (p<0.0001). <b>B</b>) ROC curve analysis for KIAA0101 mRNA expression. The receiver operating characteristic curve (ROC) is depicted on graph using RT PCR expression data of KIAA0101 normalized to GAPDH in normal adrenal cortex (n  =  21), benign adrenocortical tumors (n  =  80), ACC (n  =  10) . The area under the curve (AUC) was 0.78. A perfect diagnostic marker without any false-negatives or false-positives would have an AUC of 1. <b>C</b>) KIAA0101 protein expression in ACC. KIAA0101 protein expression levels in normal adrenal cortex (n = 5), benign (n = 13), and malignant adrenocortical tumors (n = 3) as shown in representative Western blot with KIAA0101 specific and β-Actin control antibodies. β-Actin signal was used as a control for protein loading. C  =  ACC, B  =  benign adrenocortical tumor, and N  =  normal adrenal cortex. <b>D</b>) Scatter plot of KIAA0101 mRNA and protein expression level in normal, benign and malignant adrenocortical tissue samples. Y-axis shows normalized mRNA expression by quantitative RT-PCR and X-axis shows protein expression level in the same sample based on band densitometry measurement normalized to β actin expression level. Spearmen correlation coefficient <b>(r)  = 0.61, p<0.001. </b><b>E</b>) Immunohistochemistry for KIAA0101 protein expression in normal adrenocortical tissue, benign tumors and ACC. Representative images are shown for each category at 20X magnification. Arrow indicates the positive nuclear staining for KIAA0101. In normal tissue samples, only the subcupsular region was positive. <b>F</b>) KIAA0101 cellular localization was analyzed by immunoflouroscence. Nuclei were stained by DAPI (blue), red color indicates KIAA0101 expression. Representative images from malignant ACC and normal samples are shown at 20X magnification.</p

Genomic PCR Primer sequences for KIAA0101 exons.

<p>Genomic PCR Primer sequences for KIAA0101 exons.</p

Cell proliferation increased with KIAA0101 siRNA knockdown in the NCI-H295R cell line.

<p><b>A</b>) The number of cells for the KIAA0101 siRNA treated and negative control treated groups are shown at indicated time points (significant values are indicated by asterisk (*) (p  =  0.005; relative to negative control). Error bars represent ± standard error of mean and is representative of four experiments. NC80 = Negative control at 80 nM; SI80  = siRNA at 80 nM. <b>B</b>) KIAA0101 gene knockdown and cell cycle analysis is indicated at different time points. Representative image of cell cycle profile from siRNA and negative control treatment groups. <b>C</b>) Each column indicates the distribution of G0/G1 population of cells in each group at various time points. Error bars represent ± standard error of mean and is representative of four experiments.*Day 3 and 5, p<0.05; relative to negative control.</p