Tigate the relationship amongst POSTN and STAT1 activation in vivo, sections from subcutaneous ESCC xenograft2013 Macmillan Publishers LimitedhT ERTp5R-PROSTNhT ER -P T-p O 53 ST NT-n p53 eoR17 5HPeriostin and tumor invasion GS Wong et alEPC-hTERT-p53R273H-POSTN EPC-hTERT-p53R273H-neo -POSTN -neoV143AV143AEPC-hTERT-pEPC-hTERT-pLysates 37 32 Automobile Automobile 5 Fold Modify four 3 two 1h p5 TE 3 R RT ne 273H o h p5 TE PO three R27RT ST 3H N h p5 TE 3 V1 RT ne 43A o h p5 TE PO three V14RT ST 3A NInvasionInvasion*Fold Change5 four three 2 1 0 hTERTV143A -neo p53 hTERTp53V143A-POSTN5-ID (M) 0.5 15-ID (M) 0.five 1 5 POSTN p21 GAPDH*POSTN -actin Lysates POSTN Conditioned media*Conditioned media POSTN EPC-hTERTR175H p53 neo EPC-hTERTp53R175HPOSTN1.five Fold Adjust in invasionEPC-hTERT-p53R175H-POSTNEPC-hTERT-p53R175H-POSTN Automobile 5-ID (3 M) 1.5 Fold Alter Invasion in organotypic culture1.1.0.Taurine 0.*0.0 Automobile 5-ID0.0 Vehicle 5-IDFigure three. Restoration of wild-type p53 signaling decreases POSTN expression and invasion into ECM. (a) Western blot confirming POSTN expression in EPC-hTERT-p53R273H and EPC-hTERT-p53V143A cell lines and conditioned media. pFB neo was employed as an empty manage vector. (b) Transwell Boyden Chamber invasion assay showing increase in invasion in EPC-hTERT- p53R273H and mutant p53 temperature-sensitive EPC-hTERT- p53V143A cells overexpressing POSTN compared with handle neo cells. Bar graphs represent fold adjustments .e.m. *Po0.003 (Student’s t-test, EPC-hTERT-p53V143A-POSTN cells vs manage cells). Note that Po0.05 is statistically important. Experiments were completed in triplicate. (c) Transwell Boyden Chamber invasion assay shows decrease in invasion in EPC-hTERT- p53V143A-POSTN cells when wild-type p53 conformation is induced at permissive temperature 32 1C compared with mutant p53 conformation at 37 1C. Bar graphs represent fold adjustments .e.m. *Po0.003 (Student’s t-test, EPC-hTERT-p53V143A-POSTN cells vs manage cells at 37 1C).Tirofiban Experiments were performed in triplicate.PMID:24507727 (d) Western blot evaluation of POSTN expression in EPC-hTERT- p53R175H-POSTN and EPC-hTERT- p53R175H-neo cell lysates and conditioned media soon after 24 h treatment with 5-ID (Car, 0.five mM, 1 mM and five mM). Immunoblotting for p21 to indicate restoration of wild-type p53 signaling. GAPDH was applied as a loading manage. (e) Transwell Boyden Chamber invasion assay shows decrease in invasion in EPC-hTERTp53R175H-POSTN cells following 24 h treatment of 5-ID (3 mM). Bar graphs represent fold modifications. Experiments were performed in triplicate. (f ) Hematoxylin and eosin staining of organotypic cultures comparing EPC-hTERT- p53R175H-POSTN cells treated with car and 5-ID (three mM) and show decreased invasion into the ECM immediately after remedy. Bar graphs represent fold changes. Bar one hundred mM and represent .e.m. *Po0.04 (Student’s t-test, EPC-hTERT-p53R175H-POSTN cells, treated with 5-ID vs vehicle-treated cells). Experiments were performed in triplicate.tumors (Figures 1a and b) were examined for phospho-STAT1 (Tyr701) by immunohistochemistry. Interestingly, we observed decreased nuclear STAT1 phosphorylation both in ESCC xenograft tumor cells and stroma with induction of POSTN knockdown by doxycycline (Figures 6a and b). Additionally, lysates from these xenograft tumors were analyzed, and we noted that POSTN knockdown in these tumors resulted in decreased STAT1 expression, a concomitant lower in p53 expression along with a lower in downstream STAT1-related genes (Figures 6c and d; Supplementary Figure S8). Collecti.