Expression of ERα and PRSS23 in human breast carcinoma.

<p>Immunohistochemical analysis revealed expression level of ERα (<b>A</b>, <b>B</b>, <b>C</b>, <b>D</b>, <b>E</b>, <b>F</b>) and the corresponding PRSS23 expression of the same sample (<b>G</b>, <b>H</b>, <b>I</b>, <b>J</b>, <b>K</b>, <b>L</b>) in 6 different breast cancer specimens. The scale bar is 50 µm.</p

Expression analyses of PRSS23 in human cell lines.

<p>Expression levels of PRSS23 as well as ERα were analyzed in eight different cell lines: MCF-7, BT-474, Hs.578t, MDA-MB-231, T-47D (all breast cancer), MCF-10A (mammary epithelial), RL95-2 (endometrial cancer), and Ca-SKi (cervical cancer) cell lines. <b>A.</b> Immunoblot analysis showed protein expression level of ERα in these human cell lines. <b>B.</b> qRT-PCR analysis showed relative gene expression of PRSS23 mRNA level. <b>C.</b> Immunoblot analysis showed protein expression level of PRSS23 and GAPDH in these human cell lines. The cell lysate was loaded 20 µg protein for each well in immunoblot anaylsis. qRT-PCR was performed in duplicate.</p

ERα upregulated PRSS23 expression through its upstream promoter region at −2029 to −342 bp in MCF-7 cells.

<p><b>A.</b> This scheme depicts the <i>p</i>GL3-basic constructs containing the truncated <i>PRSS23</i> promoters. Hormone-starved MCF-7 cells were separately transfected with the constructs for 12 h in phenol-red-free medium containing 10% CDS-FBS. Transfected MCF-7 cells were treated with 10 nM E₂ or vehicle control (250 ppm ethanol) for 16 h in phenol-red-free medium containing 10% CDS-FBS. Level of relative luciferase units (RLUs) were normalized to ethanol control. * <i>p</i><0.05 and ** <i>p</i><0.01 by the Mann-Whitney U test. <b>B.</b> Hormone-starved MCF-7 cells were treated with vehicle control (250 ppm ethanol) or 10 nM E₂ for 60 min. The binding of ERα to the upstream promoter region of the <i>PRSS23</i> gene and the promoter of <i>pS2</i> gene was examined in a ChIP assay. Input control was 10% of original input cell lysate. NRS stands for nonspecific rabbit serum. These results are representative of three individual experiments.</p

Gene expression analysis of breast cancer patients.

<p><b>A.</b> Clustering of self-organizing maps was done to analyze gene expression of proteases, ESR1 and ESR1-coregulated genes among 90 breast cancer patients. The red-colored boxes represent upregulated genes (ratio of log₁₀ intensity), and the green-colored boxes indicate downregulated genes. The cluster to the left shows the hierarchy relationship of gene expression patterns, and the cluster at the top indicates correlation among groups of patient samples. The lowest box represents corresponding immunohistochemistry results of ERα staining for each sample (open is positive, and filled is negative). <b>B.</b> The box plot showed expression intensity of PRSS23, CTSF, CTSC, and MMP24 in 52 ERα-positive breast cancer specimens.</p

E₂-activated ERα enhances PRSS23 expression in MCF-7 cells.

<p><b>A.</b> MCF-7 cells were treated with 1 nM E₂, 25 ppm ethanol, 5 µM Tam, and 0.5% dimethyl sulfoxide (DMSO) in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. The bar plots depicted the results of time-lapse profiling of PRSS23 mRNA levels at 6, 12, and 24 h. All experiments were performed in triplicate. The bars represent relative expression levels of PRSS23 after treatment, which was normalized to the level of 6 h-treated cells (mean ± S.E.M.). <b>B.</b> MDA-MB-231 cells were treated with 1 nM E₂ in phenol-red-free culture medium containing 10% CDS-FBS for 24 h. Expression of PRSS23 (upper panel) and pS2 (lower panel) was evaluated by qRT-PCR at 0, 6, 12, and 24 h. The bars represented the gene expression levels of PRSS23 after treatment, which was normalized to the level of untreated cells (mean ± S.E.M.).</p

PRSS23 knockdown reduced estrogen-driven MCF-7 cell proliferation.

<p><b>A.</b> The PRSS23 knockdown efficacy in MCF7 cells treated with nonspecific control (NSC) or PRSS23-specific RNAi was validated by immunoblotting. GAPDH was used as the loading control. The bar chart shows the normalized protein level of PRSS23 in NSC and PRSS23 RNAi cells. <b>B.</b> The tumor sphere formation abilities of cells were evaluated in the soft-agar tumor formation assay in the presence or absence of PRSS23 RNAi. The upper panel shows a representative picture of tumor sphere formation in 0.4% soft-agar (scale bar is 200 µm). The bar chart shows that normalized diameter of examined tumors (n≥50). The results are the average of two individual experiments. <b>C.</b> After culturing in phenol-red-free medium containing 0.5% CDS-FBS for 48 h, the cells were stimulated with 20% CDS-FBS and 1 nM E₂ or 25 ppm ethanol for 24 h. The table shows the DNA distribution profile of the examined cells. Each value is the average count of the cells in three individual experiments. * <i>p</i><0.05 and ** <i>p</i><0.01 by the Mann-Whitney U test.</p