The decreased expression of Snail by IL-27 was not reversed by inhibition of STAT3 activation. The mechanism driving the differential effect of IL-27 on the two mesenchymal markers MS 275 (N-cadherin and Vimentin) is unclear as selective inhibition of STAT1 or STAT3 did not elucidate a clear mechanism (Figure 4). Instead, there was suggestion that STAT3 may be involved in N-cadherin expression (Figure 4). Although N-cadherin is considered a mesenchymal marker, its function may be more 3-deazaneplanocin A mouse complex as other studies have shown that repression

of N-cadherin is required for epithelial to mesenchymal transition in some instances such as neural crest migration [34, 38]. However, the overall effect BIBW2992 research buy with IL-27 stimulation in our study was promotion of mesenchymal to epithelial transition. The impact of N-cadherin and STAT3 in this process is unclear. Overall, these results suggest that the STAT3 pathway is not critically involved in the IL-27 mediated promotion of epithelial marker expression. In summary, STAT1 appears to be the dominant pathway by which IL-27 promotes the expression of epithelial markers. Of note, the reciprocal increase in P-STAT3 compared to control with inhibition of STAT1 by siRNA seen in Figure 3A

is not demonstrated in Figure 4. These are two different experiments where the duration of IL-27 stimulation and time point for measurement of P-STAT3 expression are entirely different for the two figures. IL-27 inhibition of in vitro cell migration is mediated by a STAT3-independent and STAT1-dependent pathway To further evaluate phenotypic changes associated with IL-27- epithelial marker expression beyond morphologic appearance, we examined in vitro cell migration, a defining feature of the mesenchymal phenotype, by creating a scratch or wound in a confluent monolayer of NSCLC cells and evaluating wound closure as

a result of cell migration. Borders of the Thymidine kinase wound were marked by solid black lines. We expected IL-27 to inhibit cell migration through STAT1 pathway. Indeed, A549 cells treated with IL-27 showed only poor migration into the border line (lower right, Figure 5A) whereas untreated cells displayed rapid migration after 24 hours of IL-27 treatment (lower left, Figure 5A). Next, we examined whether the inhibitory effect of IL-27 on migration is related to STAT pathways using STAT1 siRNA and STAT3 inhibitor, Stattic. Again, whereas untreated cells demonstrated rapid cell migration toward each other with partial closing of the gap between the solid black lines (upper left, Figure 5B), IL-27 treated cells showed remarkably decreased cell migration (upper right, Figure 5B). Pretreated cells with STAT1 siRNA showed no significant difference in cell migration as compared to untreated cells (lower left, Figure 5B).