Figure 4. STAT3 phosphorylation is mediated by the tyrosine kinase JAK2 in NSCLC.

A, The indicated NSCLC lines were plated at a fixed density and allowed to adhere overnight. Cells were then treated for 16 hours with DMSO (con) or a 2-fold escalating dose of the JAK1/2 inhibitor ruxolitinib ranging from 0.25 to 4.0 µM (left-right). Protein lysates were harvested and the phosphorylation status of STAT3 (pSTAT3^Y705), AKT (pAKT^S473), ERK (pERK^T202/Y204), and S6 (pS6^S235/S236) was evaluated by immunoblot. Equal amounts of protein (20 µg) were added for each sample. B, The same NSCLC lines were plated as in A, but were treated with a fixed dose of ruxolitinib (1 µM) for the indicated time. Phospho-protein status was evaluated in 20 µg of protein per sample as in A. C, NCI-H1703 cells were infected with lentiviral vectors containing a control shRNA (NT, non-targeting) or one of two shRNAs targeted to human JAK2 (sh1 or sh2). Protein lysates were harvested and evaluated for JAK2 expression and STAT3 phosphorylation by immunoblot. Blots for total STAT3 and tubulin were included to demonstrate equal loading. D, Representative images of immunohistochemical stains for total JAK2, total STAT3 and phosphorylated STAT3 (pSTAT3^Y705) performed on a tissue microarray containing 245 pathologically verified human NSCLC samples. Images were captured from staining performed serial sections of the same tumor samples. E, Summary of staining results for JAK2, STAT3 and pSTAT3^Y705 on the NSCLC tissue microarray (n = 245). Note that all but one of the samples with positive nuclear pSTAT3^Y705 staining (52/53, 98.1%) were also positive for JAK2.