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

<p><b>A</b>, 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>B</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. <b>C</b>, 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. <b>D</b>, 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. <b>E</b>, Summary of staining results for JAK2, STAT3 and pSTAT3^Y705 on the NSCLC tissue microarray (<i>n</i> = 245). Note that all but one of the samples with positive nuclear pSTAT3^Y705 staining (52/53, 98.1%) were also positive for JAK2.</p

Erlotinib fails to inhibit STAT3 activation in EGFR mutant NSCLC cell lines.

<p><b>A–B</b>, Protein lysates from HCC827 (A) and HCC4006 (B) cells treated with DMSO (vehicle) or 2 µM erlotinib for 8 hours were analyzed with antibody microarrays to detect phosphorylation status of receptor tyrosine kinases and key cell signaling proteins. Fluorescent signals for the indicated spots were background subtracted and normalized to positive control spots (indicated with +) on the array. Data is shown as the percentage of average fluorescence for each set of duplicate spots relative to the average fluorescence of ten positive control spots for each array. Data on each graph is ordered according to the number (1–10) indicated on each array. <b>C</b>, HCC827 and HCC4006 cells were treated for one hour with DMSO (con) or a 2-fold escalating dose of erlotinib ranging from 0.25 to 1.0 µM (left-right). The phosphorylation status of EGFR (pEGFR^Y1068), 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>D</b>, HCC4006 cells were treated for two hours with DMSO or 1.0 µM erlotinib, then fixed and stained with antibodies for total STAT3. Cells were counter-stained with DAPI to indicate nuclear accumulation of STAT3. Scale bars, 20 µm.</p

JAK2 inhibition with ruxolitinib inhibits NSCLC cell growth in soft agar and in xenograft assays.

<p><b>A–B</b>, NSCLC cells were plated into a soft-agar suspension (5,000 cells/35 mm well) and overlaid with normal media containing 10% FBS +/− 2.0 µM ruxolitinib. After one month, agar plugs were fixed and stained with crystal violet to identify individual colonies. Digital images of each well were captured and analyzed for colony number and size. Average colony formation efficiency ([# colonies/# plated cells] ×100%, <b>A</b>) and mean colony size (area in µm², <b>B</b>) for triplicate experiments are shown. Error bars indicate the standard deviation for triplicate wells. (*, <i>p-value</i><0.05). <b>C</b>, HCC827 cells were xenografted into the flanks of nude mice. When tumors reached a volume of 100 mm³, mice were separated into two treatment arms (<i>n</i> = 6, each) and dosed daily by oral gavage with vehicle or the JAK2 inhibitor ruxolitinib (50 mg/kg). Tumor size was evaluated every other day. Animals were sacrificed when tumors reached 500 mm³ or after one month of treatment. Error bars indicate the standard error of tumor volume measurements. (*, <i>p-value</i><0.05).</p

JAK2 inhibition with ruxolitinib does not affect kinase inhibitor toxicity in two-dimensional tissue culture.

<p><b>A</b>, NCI-H358 cells (KRAS mutant) were plated at fixed density and treated for 24 hours with DMSO (−), U0126 (5.0 µM), ruxolitinib (1.0 µM) or both drugs. 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>B</b>, NCI-H358 cells were treated in quadruplicate with a 3-fold serial dilution of the MEK1/2 inhibitor U0126 (range, 0–30 µM) +/− 1.0 µM ruxolitinib. Cell viability was measured 24 hours after treatment. Averaged values for each condition are shown as a percentage of vehicle-treated (DMSO) cells. Error bars indicate standard deviations of the four replicate values. <b>C</b>, HCC4006 cells (EGFR mutant) were plated at fixed density and treated for 24 hours with DMSO (−), erlotinib (1.0 µM), ruxolitinib (1.0 µM) or both drugs. Protein lysates were harvested and analyzed as in A. <b>D</b>, HCC4006 cells were treated in quadruplicate for 24 hours with a 3-fold serial dilution of the EGFR inhibitor erlotinib (range, 0–1.0 µM) +/− 1.0 µM ruxolitinib. Cell viability and percent survival was evaluated as in B. <b>E</b>, NCI-H1703 cells (PDGFR amplified/mutant) were plated at fixed density and treated for 24 hours with DMSO (−), sunitinib (1.0 µM), ruxolitinib (1.0 µM) or both drugs. Protein lysates were harvested and analyzed as in A. <b>F</b>, NCI-H1703 cells were treated in quadruplicate for 24 hours with a 3-fold serial dilution of the PDGFR inhibitor sunitinib (range, 0–100 µM) +/− 1.0 µM ruxolitinib. Cell viability and percent survival was evaluated as in B.</p

Targeted kinase inhibitors fail to downregulate STAT3 phosphorylation in NSCLC cell lines with diverse driver mutations.

<p><b>A</b>, KRAS mutant NSCLC cell lines (A549, NCI-H358 and NCI-H460) were treated for one hour with DMSO (con) or a 2-fold escalating dose of the MEK1/2 inhibitor U0126 ranging from 0.625 to 5.0 µM (left-right). 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>B</b>, PDGFRA amplified/mutant NSCLC cells (NCI-H1703) were treated for one hour with DMSO (con) or a 2-fold escalating dose of the PDGFRA inhibitor sunitinib ranging from 0.625 to 2.5 µM (left-right). Phospho-protein status was evaluated in 20 µg of protein per sample as in A. <b>C</b>, MET amplified NSCLC cells (NCI-H1993) were treated for one hour with DMSO (con) or a 2-fold escalating dose of the MET/ALK inhibitor crizotinib ranging from 0.25 to 1.0 µM (left-right). Phospho-protein status was evaluated in 20 µg of protein per sample as in A. <b>D</b>, The indicated NSCLC lines were plated at fixed densities and allowed to adhere for 24 hours. Cells were then treated for 18 hours in normal media with (+) or without (−) 10% fetal bovine serum (FBS). Phospho-protein status was evaluated in 20 µg of protein per sample as in A. Even sample loading is indicated by replicate immunoblot for beta-tubulin.</p

STAT3 phosphorylation is mediated by IL-6 family cytokines in NSCLC cell lines.

<p><b>A</b>, The indicated NSCLC lines were plated at fixed densities and allowed to adhere overnight in serum-containing media. Cells were then treated for 24 hours in serum-free media containing anti-gp130 neutralizing antibody or a control mouse IgG (2.0 µg/mL each). 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 (30 µg) were added for each sample. <b>B</b>, NSCLC lines were plated at fixed densities and allowed to adhere overnight in serum-containing media. Cells were then serum-starved, and conditioned media was collected at 48 hours. Secreted interleukin-6 (IL-6) levels were measured from the indicated cell lines using a bead-based immunoassay platform. Values shown are averages of duplicate measurements for each sample.</p

Comparison between pyrosequencing and culture for detecting the nine bacterial families that were successfully cultured at least once, among all wound samples (n = 32).

<p>Comparison between pyrosequencing and culture for detecting the nine bacterial families that were successfully cultured at least once, among all wound samples (n = 32).</p

Comparison of indicator species prevalence (out of n = 300 sequences for each sample) between untreated and antibiotic treated wounds.

*<p>The empirical p-values comparing the prevalence of indicator species between the two antibiotic use groups were generated using the Monte Carlo method. The statistical significance level after the Bonferroni correction was 0.05/3 = 0.017.</p>**<p>The increase in <i>Pseudomonadaceae</i> in the antibiotic treated group was significant at <i>p</i> = 0.0046<0.017.</p

The nMDS ordination plot comparing wound bacterial communities from antibiotic treated participants and untreated participants.

<p>Each data point in nMDS plot represent the bacterial community identified from a single wound specimen. Comparison using MRPP found that the antibiotic treated and untreated wound microbiota are significantly different (<i>p</i> = 0.0069).</p

Estimated complexity at different taxonomic levels by pyrosequencing and culture.

<p>Estimated complexity at different taxonomic levels by pyrosequencing and culture.</p