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We also examined the frequency of primary cilia on breast cancer cells by staining tumor samples from 39 patients with pre-malignant carcinoma in situ Entinostat research buy (CIS) and from 65 patients with invasive breast carcinoma (INV). The first serial section was stained with H&E to identify areas of normal, CIS, and INV. The H&E slide was used as a reference to find the tissue types of interest on the adjacent serial section, which was stained with antibodies recognizing acetylated tubulin (Ac-Tub) to visualize primary cilia and ��-tubulin (��-Tub) to identify their associated centrosomes (Figure?1A and ?and1B).1B). Acetylated tubulin was validated for use as a marker of primary cilia in these tissue samples by co-staining with an antibody that recognizes Arl13b, a well characterized marker of primary cilia (Additional file 1: Figure S1). Ac-Tub and Arl13b were observed to co-localize in cilia found on normal and cancer tissues. The normal mammary gland is composed of basal and luminal epithelial cells that are separated from surrounding stromal cells by a basement membrane. Basal cells in the mammary gland include both myoepithilial cells and suprabasal cells [19]. We previously reported that basal epithelial cells in the developing normal murine mammary gland have a higher frequency of ciliated cells compared to luminal epithelial cells [20]. For our current study, we used basal position Reelin of epithelial cells as well as co-staining with antibodies specific for basal cells including ��-Smooth Muscle Actin (��-SMA) PLX 4720 and p63 to determine the frequency of cilia on basal versus luminal epithelial cells in histologically normal human tissue (Additional file 1: Figure S2). While primary cilia were expressed on both basal and luminal epithelial cells, basal epithelial cells had a higher cilia frequency (Basal: RM, median=23.6%; HNE, median=22.3%) than luminal epithelial cells (Luminal: RM, median=1.1%; HNE, median=0.5%) (Figure?1A, ?A,1C,1C, and ?and1D).1D). These results demonstrate that both basal and luminal epithelial cells have cilia-positive cells and that the frequency is higher for basal cells. Invasive carcinoma (INV) of the breast is classified into three grades (INV: I, 2, and 3) of increasing atypical tissue and representing increased aggressive potential of the tumor. INV-1 and INV-2 are often grouped together as low-grade and INV-3 is considered high-grade. We quantified cilia frequency on INV cancer cells and observed a decrease in the percentage of ciliated cancer cells in INV-1, INV-2, and INV-3 (Figure?1B, ?B,1C,1C, 1D). The median percentage of ciliated cells in INV-1 and INV-2 when combined (median=0.2%; P