Fig 1: Expression of CHST11, CHST3 and CHST7 in epithelium and stroma.(A) In the normal and malignant prostate stromal and epithelial tissues obtained by laser capture -microdissection (LCM), mRNA expression of the chondroitin-4-sulfotransferase CHST11 is significantly greater in the stroma, both normal and malignant, than in the epithelium (p < 0.001, n = 6). CHST expression is significantly lower in the malignant epithelium, compared to the normal epithelium (p < 0.001; n = 6). (B) In cultured normal prostate stromal cells, CHST11 expression is significantly more than in epithelial cells (p < 0.001; n = 6). ARSB silencing by siRNA leads to declines in CHST11 expression in both stromal and epithelial cells (p < 0.001; n = 6). (C) In the normal and malignant prostate stromal and epithelial tissues obtained by LCM, the mRNA expression of CHST3, a chondroitin-6-sulfotransferase, is higher in the epithelium than in the stroma, and similar in normal and malignant tissue (p < 0.001, n = 6). CHST7, another chondroitin 6-sulfotransferase, is not significantly different in stroma vs. epithelium or in malignant vs. normal tissue. (D) There are no significant differences in CHST7 expression between the prostate stromal and epithelial cells when ARSB is silenced. (E) The expression of CHST11 is increased in both the normal human prostate epithelial cells (CRL-2850) and in the malignant PC-3 cell line (CRL-1435, ATCC) following exposure to exogenous TGF-β (10 ng/ml × 24 h; p < 0.001, n = 3). This is consistent with previous data about the effect of TGF-β on CHST11 expression and shows that the pathway for expression is intact in the epithelial cells [50]. *** represents p ≤ 0.001 and greater than control; ### represents p ≤ 0.001 and less than control. [ARSB = arylsulfatase B; CHST = chondroitin sulfotransferase; LCM = laser-capture microdissection; PC-3 = metastatic prostate cell line; PEC = prostate epithelial cell; si = siRNA; TGF-β = transforming growth factor β]
Fig 2: Chondroitin sulfotransferase (CHST) 15 (chondroitin 4-sulfate 6-O-sulfotransferase) is increased in malignant prostate epithelial tissue, in prostate tissue of ARSB-null mice, and in prostate epithelial cells when ARSB is reduced.(A) In laser-microdissected normal and malignant human prostate epithelium and stroma, CHST15 mRNA expression is increased in the malignant epithelial tissue compared to normal epithelial tissue (p < 0.001; n = 6). In the normal and malignant stroma, CHST15 expression is less than in the normal epithelium (p < 0.05; n = 6). (B) In the laser-microdissected prostate tissues, CHST15 protein detected by ELISA was significantly greater in the malignant prostate tissue (p < 0.001; n = 3). Stromal values are significantly less than in the normal epithelial tissue (p < 0.05; n = 3). (C) In prostate tissues from ARSB-null mice (Strain 005598, Jackson Labs), the CHST15 mRNA was significantly more than in the prostate tissue from normal C57BL/6J controls (p < 0.001; n = 6). (D) In cultured human prostate epithelial cells (CRL-2850, ATCC) treated with prostate stromal cell (CRL-2854, ATCC) spent media in 1:1 ratio with epithelial cell media, CHST15 expression increased following ARSB silencing by siRNA in the epithelial cells (p < 0.001; n = 6). Expression was significantly higher in the epithelial cells treated with control siRNA than in the stromal cells (p < 0.01; n = 6). (E) Correspondingly, the CHST15 protein determined by ELISA was significantly greater in the epithelial cells grown with spent media from the stromal cells in 1:1 combination with epithelial cell media and ARSB silencing by siRNA (p < 0.001; n = 3). [ARSB = arylsulfatase B; CHST = chondroitin sulfotransferase; SCM = prostate stromal cell spent media; si = siRNA; *** for p < 0.001 greater than control; ## for p < 0.01 and # for p < 0.05 less than control]
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