E periments were re peated at least 3 times in duplicate. Proliferation assay HTR8 SVneo cells were plated in 96 well plate in a final volume of 100 ul well culture medium in the absence or presence of OSM and stattic. Cells were in cubated for 12 h and 48 h. After adding 10 ul of water namely soluble tetrazolium reagent to each well, cells were incubated for 4 h in standard culture conditions. The absorbance of the samples was measured using a 96 well plate reader at 450 nm. The E G reference wavelength was 650 nm. E periments were re peated at least 3 times in duplicate. Statistical analysis Data are e pressed as mean SEM. The non parametric Mann Whitney rank sum test and an independent t test were used to compare the 2 groups. A p value of 0. 05 or less was considered to be statistically significant.

Each e periment was performed 3 times. Results Effects of OSM on mRNA and protein e pression of E cadherin in HTR8 SVneo cells OSM significantly reduced E cadherin RNA and protein e pression, compared to the control group, after 48 h stimulation. STAT3 phosphorylation is stimulated by OSM in HTR8 SVneo cells Basal levels of STAT3 phosphorylation were very low, although stimulation with OSM led to immediate and transient increases in phosphorylation. Effect of stattic on OSM mediated changes in E cadherin e pression in HTR8 SVneo cells To investigate the role of the STAT3 pathway in the OSM induced downregulation of E cadherin, HTR8 SVneo cells were pretreated with stattic, which has been reported to inhibit the phosphorylation of STAT3, and then stimulated with OSM.

In western blot ting, the e pression of E cadherin, which was suppressed by OSM, at 48 h, was restored by stattic pretreatment re gardless of the concentration used. Effect of STAT3 siRNA on OSM mediated changes in E cadherin e pression in HTR8 SVneo cells Applying the described siRNA method and oligonucleotide sequence, the cellular contents of STAT3 and phosphory lated STAT3 were significantly decreased in HTR8 SVneo cells when 25 nM relevant oligos, but not when scrambled oligos were used, as analyzed by western blotting. Transfection of HTR8 SVneo cells with STAT3 siRNA significantly in creased E cadherin e pression which was suppressed by OSM without affecting the e pression of the GAPDH protein. Non targeted negative control siRNA did not affect the e pression of STAT3 and E cadherin e pression.

Effects of OSM and STAT3 inhibitor on E cadherin in HTR8 SVneo cells by indirect immunofluorescence staining After 48 h of incubation in the presence of OSM, Dacomitinib HTR8 SVneo cell staining revealed a downregulation of E cadherin compared with the controls. There was no specific Imatinib Sigma change in the e pression of E cadherin, with or without stattic pretreatment. E cadherin e pression after pretreatment with stattic and after 48 h incubation with OSM was similar to the e pression in unstimulated cells.