To confirm CRNN expression in xenograft tumor, IHC staining with www.selleckchem.com/products/AZD2281(Olaparib).html anti-CRNN antibody was performed in sections (5��m in thick) of paraffin-embedded xenograft tumor. RNA interfering (RNAi) CRNN-expressing clones CRNN-C2, C3 (KYSE30) or CRNN-C1 (KYSE180) were transfected with double-stranded siRNAs (Ambion, Carlsbad, CA) with lipofectamine 2000TM reagent (Invitrogen) according to the manufacturer��s instructions. Forty-eight hours after transfection, the gene-silencing effect was measured by qRT-PCR and western blot analysis, respectively. Three independent experiments were performed. Cell cycle analysis CRNN-C2/CRNN-C1 or Vec-30/Vec-180(2��105) were fixed in 70% ethanol and stained with propidium iodide, and DNA content was analyzed by Cytomics FC (Beckman Coulter, Indianapolis, IN).

Western blot analysis Western blotting was done according to the standard protocol with antibodies for GAPDH, CRNN (Santa Cruz Biotechnology, Santa Cruz, CA), P53, P21WAF1/CIP1, cyclin D1, CDK4, cyclin E, CDK2, Rb, tubulin (Cell Signaling Technology, Danvers, MA). Statistical analysis Statistical analysis was performed using SPSS standard version 13.0 software (SPSS Inc, Chicago, IL). Data were expressed as mean �� S.E.M. from at least three independent determinations. Significance of difference was analyzed using Student��s t-tests. The correlation between CRNN expression and clinicopathologic characteristics was analyzed using the Fisher��s exact test. Cum survival was calculated from the date of diagnosis to the date of cancer-related death or last follow-up.

Survival curve was assessed by the Kaplan-Meier method and compared by the log-rank test. Relative risks of cancer-related death associated with CRNN expression status and other predictor variables were estimated by univariate analysis. Multivariate survival analysis was done on all parameters that were found to be significant on univariate level using the Cox regression model. Differences were considered significant for P<0.05. Results Downregulation of CRNN is frequently detected in ESCC The mRNA expression of CRNN in 9 ESCC cell lines and 56 primary ESCC tumors and their paired non-tumorous tissues were detected by semiquantitative and qRT-PCR, respectively. The results showed that downregulation of CRNN was detected in 26/56 (46.4%) of primary ESCCs (Figure 1A) and 9/9 of ESCC cell lines (Figure 1B).

Carfilzomib Figure 1 Downregulation of CRNN in ESCC. CRNN downregulation correlates with poor outcome of ESCC To investigate the clinical significance of CRNN downregulation in esophageal carcinogenesis, CRNN expression in protein level was also studied using ESCC tissue microarray. Expression of CRNN was classified into absent (scored as 0), weak-positive (scored as 1+) and strong-positive (scored as 2+) cytoplasmic staining. Informative expression of CRNN was detected in 249 ESCC cases.