PDCD4 protein expression in miR-21 inhibitor–transfected cells after IL-6 treatment.

<p>PDCD4 protein expression was significantly higher in prostate cancer cells transfected with the miR-21-inhibitor before Il-6 treatment than in cells not transfected with the inhibitor (one-way ANOVA test: <i>F</i> value = 241.781, <i>P</i> < 0.05). Levels of PDCD4 protein expression in transfected and nontransfected cells of both cell lines were significantly different (LSD test: <i>P</i> < 0.05).</p

Effect of mir-21 inhibitor transfection on PDCD4 mRNA and protein expression in PC-3 and LNCaP cells.

<p>qRT-PCR results showing an increase in PDCD4 mRNA expression of PC-3 and LNCaP cells transfected with miR-21 inhibitor, indicating that PDCD4 mRNA was differentially expressed between the two groups. Among the miR-21–inhibited cells, PDCD4 mRNA expression in LNCaP cells was significantly higher than that in PC-3 cells. PDCD4 mRNA expression of LNCaP and PC-3 cells was downregulated after a 24-h treatment with different concentrations of IL-6. <b>(b)</b> Western blot analysis showing an increase in levels of expression of the PDCD4 protein in PC-3 and LNCaP cells transfected with the miR-21 inhibitor. Nontransfected and negative-control cells were ranked 1 through 6 in the rank test of paired samples. Levels of PDCD4 protein expression of LNCaP and PC-3 cells was downregulated after a 24-h treatment with different concentrations of IL-6.</p

PDCD4 expression after IL-6 treatment.

<p>Levels of PDCD4 mRNA expression in LNCaP and PC-3 cells were downregulated after a 24-h treatment with IL-6 at 0, 5, or 10 ng/mL. The difference there was statistical significance (<b>a</b>) between each other. The same difference was also observed in the levels of PDCD4 protein expression (<b>b</b>). <b>(a)</b> PDCD4 mRNA expression of LNCaP and PC-3 cells was downregulated after a 24-h treatment with IL-6 at 10 ng/mL. <b>(b)</b> PDCD4 protein expression of LNCaP and PC-3 cells was downregulated after a 24-h treatment with IL-6 at 10 ng/mL,</p

PDCD4 and PSA expression in prostate cancer, prostatic intraepithelial neoplasia and prostatic hyperplasia.

<p>In prostate cancer, considerable amount of normal gland is lacking. Cancer nests are formed with irregular cribriform glands, and cancer infiltrates the muscle tissue and cell cytoplasm. This staining pattern was graded PDCD4(+), PSA(+++). In prostatic intraepithelial neoplasia, the staining pattern was graded PDCD4(++), PSA (++). In prostatic hyperplasia/glandular hyperplasia, the nuclear staining pattern was graded PDCD4 (+++), PSA (+/−). The expression of PDCD4 and PSA were different (<i>x</i>² = 8.632, <i>P</i><0.05) among the 3 tissue types. In the Spearman rank test, the expression levels of PDCD4 and PSA among the 3 prostate tissue types were negatively correlated (−1 < <i>r</i> < 0).</p

Identification of Covalent Binding Sites Targeting Cysteines Based on Computational Approaches

Covalent drugs have attracted increasing attention in recent years due to good inhibitory activity and selectivity. Targeting noncatalytic cysteines with irreversible inhibitors is a powerful approach for enhancing pharmacological potency and selectivity because cysteines can form covalent bonds with inhibitors through their nucleophilic thiol groups. However, most human kinases have multiple noncatalytic cysteines within the active site; to accurately predict which cysteine is most likely to form covalent bonds is of great importance but remains a challenge when designing irreversible inhibitors. In this work, FTMap was first applied to check its ability in predicting covalent binding site defined as the region where covalent bonds are formed between cysteines and irreversible inhibitors. Results show that it has excellent performance in detecting the hot spots within the binding pocket, and its hydrogen bond interaction frequency analysis could give us some interesting instructions for identification of covalent binding cysteines. Furthermore, we proposed a simple but useful covalent fragment probing approach and showed that it successfully predicted the covalent binding site of seven targets. By adopting a distance-based method, we observed that the closer the nucleophiles of covalent warheads are to the thiol group of a cysteine, the higher the possibility that a cysteine is prone to form a covalent bond. We believe that the combination of FTMap and our distance-based covalent fragment probing method can become a useful tool in detecting the covalent binding site of these targets